Apparatus and system having dry control gene silencing compositions

ABSTRACT

An RTF testing plate can include at least a first control well including a substantially dry first control composition having at least a first control siRNA. The first control siRNA is capable of providing a first indication of the gene silencing efficacy. Additionally, the first control composition can be configured such that the first control siRNA is capable of being solubilized or suspended in an aqueous medium in an amount sufficient for transfecting cells in the first control well. The control siRNA can be any one of a transfection control siRNA, positive control siRNA, or negative control siRNA. Optionally, the total amount of control siRNA in the first control composition can be present in an amount for transfecting cells in only the first control well.

CROSS-REFERENCE TO RELATED APPLICATIONS

This United States patent application claims benefit of U.S. ProvisionalApplication Ser. No. 60/630,320, filed Nov. 22, 2004, and U.S.Provisional Application Ser. No. 60/678,165, filed May 04, 2005, both ofwhich are incorporated herein by reference.

This United States Patent Application also cross-references thefollowing United States patent applications filed herewith: AttorneyDocket No. 16542.1.1, entitled APPARATUS AND SYSTEM HAVING DRY GENESILENCING COMPOSITIONS, with Barbara Robertson, Ph.D., et al. asinventors; Attorney Docket No. 16542.1.2, entitled APPARATUS AND SYSTEMHAVING DRY GENE SILENCING POOLS, with Barbara Robertson, Ph.D., et al asinventors; and Attorney Docket No. 16542.1.4, entitled METHOD OFDETERMINING A CELLULAR RESPONSE TO A BIOLOGICAL AGENT, with BarbaraRobertson, Ph.D., et al. as inventors, wherein each is incorporatedherein by reference.

BACKGROUND OF THE INVENTION

1. The Field of the Invention

The present invention relates to an apparatus and system for use in RNAinterference. More particularly, the present invention relates to anapparatus and system that include a well plate having control siRNA.

2. The Related Technology

Recently, a natural cellular regulatory pathway was discovered that usestranscribed microRNA (“miRNA”) in order to control protein production.The miRNA includes a duplex region of sense and antisense RNA. Thisregulatory pathway uses miRNA in order to target complementary mRNA toinhibit production of the encoded protein. Accordingly, a complex seriesof proteins are involved in this RNA interfering pathway to inhibit orstop production of the proteins encoded by the mRNA. As such, theprocess is referred to as RNA interference or RNAi.

Additionally, it has been found that the RNAi pathway can be used withsynthetic dsRNA (e.g., siRNA) for silencing genes and inhibiting proteinexpression. This can allow for siRNA having specific sequences to beproduced to target complementary DNA and/or mRNA encoding a specificprotein. The siRNA can interact with the natural RNAi pathway to silencea target gene and inhibit production of the encoded polypeptide. Theability to silence a specific gene and inhibit production of the encodedprotein has been used for basic research of gene function, gene mapping,cellular pathway analysis, and other gene-related studies.

In order to induce gene silencing, the siRNA needs to be introduced intoa cell. While the most common procedures for introducing nucleic acidsinto cells has been forward transfection, reverse transfection (“RTF”)has been developed more recently and used as an alternative to forwardtransfection procedures. In certain versions of RTF protocols, a complexof lipid-nucleic acid (e.g., lipoplex) can be prepared and introducedinto the test wells of a well plate. Cells are introduced into the testwells with the lipid-nucleic acid complexes, and incubated so that thesiRNA can enter the cells. Examples of some RTF protocols can be foundin U.S. Pat. No. 5,811,274 to Palsson, U.S. Pat. No. 5,804,431 toPalsson and U.S. Pat. No. 6,544,790 to Sabatini and in U.S. PublishedApplications 2002/0006664 to Sabatini and 2003/070642 to Caldwell et al.As described in these references, RTF procedures for nucleic acidsgenerally can have fewer steps compared to traditional forwardtransfection and may offer benefits in attempting to isolate thetransfected cells to particular regions of a single surface, such as aglass slide. However, RTF procedures for siRNA have not been optimizedto the point of practical application, and improvements in genesilencing efficacy are still needed, especially for situations in whichone is experimenting with multiple different siRNAs, different genetargets or different cell lines.

Often, RTF protocols can be performed on well plates in a manner that isnot optimized or produces inaccurate and unreliable data. The lack ofoptimization can cause variations between plates, and can reduce thereliability of the data. Variations in data from a lack of optimizationor an error can produce results that appear to be related to the genesilencing obtained from the siRNA, and are difficult to detect withoutcomparing the test results to other experiments performed with the samesiRNA. Additionally, systematic variations in data that occur can arisefrom the RTF conditions, and may be related to the amount of siRNA,amount of siRNA carrier, cell density, media, temperature, or variousother factors that affect gene silencing.

Therefore, it would be advantageous to have an improved RTF protocol fortesting the efficacy of gene silencing. Additionally, it would bebeneficial to have an RTF format that uses controls to test the efficacyof gene silencing.

BRIEF SUMMARY OF THE INVENTION

Generally, embodiments of the present invention include well plates,kits, systems, and methods of using the same for testing the efficacy ofgene silencing. Accordingly, the present invention provides well plates,kits, and systems that implement an improved RTF testing protocol fordelivering control siRNA into cells to test the efficacy of genesilencing in other cells in the well plate or other well plates. Thecontrol siRNA can provide an indication of gene silencing efficacy thatcan be compared to known functionalities and standard results obtainedfrom using the control siRNA in optimal or other test conditions.

In one embodiment, the present invention can include a reversetransfection plate for testing the efficacy of gene silencing. The platecan include at least a first control well including a substantially dryfirst control composition having at least a first control siRNA. Thefirst control siRNA can be capable of providing a first indication ofthe gene silencing efficacy. Additionally, the first control compositioncan be configured such that the first control siRNA is capable of beingsolubilized or suspended in an aqueous medium in an amount sufficientfor transfecting cells in the first control well. The control siRNA canbe any one of a transfection control siRNA, positive control siRNA, ornegative control siRNA. Optionally, the total amount of control siRNA inthe first control composition can be present in an amount fortransfecting cells in only the first control well.

In one embodiment, the plate can further include at least a secondcontrol well including a substantially dry second control composition.The second control composition can include at least a second controlsiRNA, which can be any of the transfection, positive, or negativecontrol siRNAs. Preferably, the second control siRNA is different fromthe first control siRNA. As such, the second control siRNA can provide asecond indication of gene silencing efficacy that is different from thefirst indication. The second control composition can be configured suchthat the second control siRNA is capable of being solubilized orsuspended in an aqueous medium in an amount sufficient for transfectingcells in the second control well. Optionally, the first controlcomposition includes a positive control siRNA and the second controlcomposition includes a negative control siRNA. Alternatively, the firstcontrol composition includes a positive control siRNA and the secondcontrol composition includes a transfection control siRNA. In yetanother alternative, the first control composition can have atransfection control siRNA and the second control composition caninclude a negative control siRNA.

In one embodiment, the plate can further include at least a thirdcontrol well including a substantially dry third control composition.The third control composition can include at least a third controlsiRNA, which can be any of the transfection, positive, or negativecontrol siRNA. Preferably, the third control siRNA is different, fromthe first control siRNA and second control siRNA. As such, the thirdcontrol siRNA can provide a third indication of gene silencing efficacythat is different from the first indication and second indication. Thethird control composition can be configured such that the third controlsiRNA is capable of being solubilized or suspended in an aqueous mediumin an amount sufficient for transfecting cells in the third controlwell. Preferably, the first, second, and third control siRNAs include atransfection, positive, and negative control siRNA, respectively.

In one embodiment, the transfection control siRNA, positive controlsiRNA, or negative control siRNA can conform to the descriptionsprovided herein and in the incorporated references.

In one embodiment, the present invention provides a kit or system thatincludes a well plate having a control well with a substantially drycontrol composition comprised of control siRNA. Additionally, such a kitor system includes a polynucleotide carrier. The polynucleotide carriercan be a cationic lipid, polymer, lipopolymer, or the like.Additionally, the kit or system can include various solubilizingsolutions, reagents, cell culture media, and the like.

In one embodiment, the present invention includes a method of testingthe efficacy of gene silencing with control siRNA. This can includetesting the conditions used in the RTF protocol, which may be related tocell density, type of polynucleotide carrier, carrier concentration,siRNA concentration, RTF protocol, or other factors that can alter theeffectiveness for siRNA to silence genes. Additionally, such a testingmethod can include the use of any well plate consistent with theforegoing characterizations. Accordingly, an aqueous medium can be addedto a first control well in the well plate, wherein the first controlwell includes a first control siRNA. The aqueous medium can solubilizeor suspend the first control siRNA. Optionally, the aqueous medium caninclude a polynucleotide carrier such as a cationic lipid, polymer,lipopolymer, and the like, which can form a complex with the controlsiRNA. The complex can be prepared so as to be capable of beingsuspended or solubilized in the aqueous medium.

Cells can be added to the first well under conditions that permittransfection with the complex. The cells can be added in an amount ofabout 1×10³ to about 3.5×10⁴ or about 2×10³ to about 3×10⁴ cells perabout 0.3 cm² to about 0.35 cm² of cell growth surface area.Subsequently, the control siRNA can contact the cell in a manner thatallows for entry into the cellular cytoplasm. However, any mode oftransfection can be used to cause the control siRNA to enter the cell.The well plate can then be maintained under conditions so that cellgrowth, cell division, transfection, and/or gene silencing occurs. Aftera proper duration that allows for transfection and/or the control siRNAto silence a known gene, the effect of the first control siRNA on thecells can be determined. The effect of the first control siRNA in thecells can be compared with a known effect of the first control siRNA.

In one embodiment, a second control siRNA can be used to test theefficacy of gene silencing. As such, the testing protocol can includeadding the aqueous medium to a second control well in the well plate,wherein the second control well includes a second control siRNA.Subsequently, the cells can be added to the second control well underconditions that permit transfection, and the second control siRNA canthen be transfected into the cells by any mode of transfection. Theeffect of the second control siRNA on the cells can be determined. Theeffect of the second control siRNA can be compared to the effect of thefirst control siRNA.

In one embodiment, a third control siRNA can be used to test theefficacy of gene silencing. As such, the testing protocol can includeadding the aqueous medium to a third control well in the well plate,wherein the third control well includes a third control siRNA.Subsequently, the cells can be added to the third control well underconditions that permit transfection, and the third control siRNA canthen be transfected into the cells by any mode of transfection. Theeffect of the third control siRNA on the cells can be determined. Theeffect of the third control siRNA can be compared to the effect of thefirst control siRNA and/or the effect of the second control siRNA.

In one embodiment, a blank well that is substantially devoid of siRNAcan be used to test the efficacy of gene silencing. As such, the testingprotocol can include adding an aqueous medium to a blank well in thewell plate, the blank well being devoid of siRNA. Optionally, theaqueous medium can include the polynucleotide carrier. Optionally, thecells are then added to the blank well, and the effects of control siRNAon the cells are compared to the cells added to the blank well. On theother hand, the cells may not be added to the blank well so that it canbe used for various calibrations.

These and other embodiments and features of the present invention willbecome more fully apparent from the following description and appendedclaims, or may be learned by the practice of the invention as set forthhereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

To further clarify the above and other advantages and features of thepresent invention, a more particular description of the invention can berendered by reference to specific embodiments thereof which areillustrated in the appended drawings. It is appreciated that thesedrawings depict only typical embodiments of the invention and aretherefore not to be considered limiting of its scope. The invention canbe described and explained with additional specificity and detailthrough the use of the accompanying drawings.

FIGS. 1A and 1B are schematic diagrams that illustrate an embodiment ofa multi-well plate having control siRNA and test siRNA.

FIGS. 2A-2D are schematic diagrams that illustrate embodiments ofarrangements of control siRNA on a multi-well plate

FIGS. 3A-3J are graphical representations of embodiments of the effectsof various plate conditions on siRNA RTF gene silencing and cellviability. FIG. 3A illustrates cell viability and FIG. 3B illustratesgene silencing of plain plates. FIG. 3C illustrates cell viability andFIG. 3D illustrates gene silencing of poly-L-lysine (“PLL”) coatedplates. FIG. 3E illustrates cell viability and FIG. 3F illustrates genesilencing of CELLBIND™ plates. FIG. 3G illustrates cell viability andFIG. 6H illustrates gene silencing of MATRIGEL™ plates. FIG. 3Iillustrates cell viability and FIG. 3J illustrates gene silencing offibronectin plates. In cell survival measurements, the Y-axis representsrelative levels of survival with 1.0 being 100% viability. In genesilencing measurements, the Y-axis represents the level of geneexpression compared to controls with 1.0 being 100% expression, and “ug”is microgram.

FIG. 4 is a graphical representation of an embodiment of a comparison ofhuman cyclophilin B gene silencing at different cell plating densities.HeLa cells at 10K, 20K, and 40K cells per well were transfected withcyclo 3, cyclo 28, or cyclo 37 siRNA at varying concentrations (e.g., 4nM-250 nM) using DharmaFECT™ 1 lipid at 0.063 micrograms (“ug”) for 10Kcells, 0.125 ug for 20K cells, and 0.250 ug for 40K cells of per 100microliters (“uL”) volume, respectively. The total volume in each wellis 125 uL. The figure demonstrates the role that cell density plays insiRNA silencing efficiency in a reverse transfection format. For genesilencing, the Y-axis represents the level of gene expression comparedto controls with 1.0 being 100% expression.

FIGS. 5A-5C are graphical representations of an embodiment of theidentification of toxic siRNA. HeLa cells were forward transfected at5,000 cells per well with 10 nM siRNA. FIG. 5A depicts a DBI-siRNA walkidentifying toxic siRNA, wherein the black bars represent DBI silencing,and the gray bars represent cell survival. FIG. 5B depicts cell survivalresulting from the introduction of one of forty-eight different siRNAdirected against one of twelve different targets. FIG. 5C depicts anexamination of eight siRNA derived from FIG. 5B, and shows that toxicityis unrelated to target specific silencing. Also, the data demonstratesthat pooling is one means of eliminating siRNA-induced toxicity.

FIGS. 6A-6C are graphical representations of an embodiment of theidentification of toxic motifs responsible for siRNA induced celltoxicity. FIG. 6A depicts thirty-eight randomly selected siRNAcontaining AAA/UUU motifs tested for the ability to induce toxicity.FIG. 6B depicts nineteen randomly selected siRNA carrying the GCCA/UGGCmotif tested for the ability to induce toxicity. FIG. 6C depictsthirty-two randomly selected siRNA that do not carry either the AAA/UUUor GCCA/UGGC motifs were tested for toxicity. In the figures, black barsrepresent sequences that induce toxicity, and gray bars representnon-toxic sequences.

FIG. 7A is a schematic representation of embodiments of assays to studythe involvement of the RNAi pathway in siRNA induced toxicity, whichshows control and experimental studies.

FIGS. 7B-7I are images of embodiments of green protein fluorescenceresulting from the control and experimental conditions demonstratingthat Ago2 silencing prevents siRNA targeting EGFP from silencing theintended target. FIG. 7B depicts EGFP expression pattern and FIG. 7Cdepicts Hoechst 33342 stained cells to show that treatment of cells witha control siRNA (e.g., siRNA-RISC-Free) in both transfection 1 (T1) andtransfection 2 (T2) does not affect EGFP expression. FIGS. 7D and 7Eshow when T1 is a control siRNA and T2 is an siRNA directed againstEGFP, it is possible to silence EGFP expression. FIGS. 7F and 7G showwhen T1 uses an siRNA directed against the eIF2C2 protein (e.g., Ago2),but T2 uses an control siRNA, EGFP expression is maintained. FIGS. 7Hand 7I show when T1 uses an siRNA directed against the eIF2C2 protein,but T2 uses an EGFP-siRNA, EGFP expression is not affected due to thedisruption of the RNAi pathway.

FIG. 7J is a graphical representation of an embodiment of testing toxicsiRNA in cells that have an intact RNAi pathway with control siRNA and adisrupted RNAi pathway with siRNA silencing eIF2C2/Ago2.

FIG. 8 is a graphical representation of an embodiment of the effects oftruncating toxic siRNA on cell viability.

FIG. 9A is a graphical representation of an embodiment of the cellviability of OLIGOFECTAMINE™, DharmaFECT™ 1 (“DF1”), and TBio in A549Cells.

FIG. 9B is a graphical representation of an embodiment of the genesilencing of the conditions of FIG. 9A.

FIG. 10A is a graphical representation of an embodiment of the cellviability of OLIGOFECTAMINE™, DharmaFECT™ 1 (“DF1”), and TBio in 3T3L1Cells.

FIG. 10B is a graphical representation of an embodiment of the genesilencing of the conditions of FIG. 10A.

FIGS. 11A and 11B are graphical representations of an embodiment of thegene silencing (FIG. 11A) and cell viability (FIG. 11B) using threedifferent media/buffer rehydration solutions, Opti-MEM™, HyQ-MEM™, andHBSS, in a reverse transfection format using DharmaFECT™ 1 (“DF1”). Thenumbers “1”, “2”, “3”, “4”, “5” and “8” refer to various biologicalreplicates performed on different days in this study.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Generally, the present invention is related to an apparatus and systemfor use in testing the efficacy of gene silencing in cells. Theapparatus includes plates with wells that have dry control compositionscomprised of control siRNA, which can be solubilized or suspended foruse in RTF testing protocols. The systems, which can be provided askits, include the plates and polynucleotide carriers that can becombined with the control siRNA to form a transfection complex capableof entering a cell in order to deliver the control siRNA. Additionally,the system or kits can include various other solutions and reagents forimplementing RTF protocols.

The well plates, systems, kits, and methods of the present invention canbe configured for use in high content screening (“HCS”) and highthroughput screening (“HTS”) applications with or without the use oflaboratory automation equipment. Also, the well plates, systems, kits,and methods can also be used with automated systems, such as roboticsystems. However, the well plates, systems, kits, and methods can alsobe used in RTF testing protocols without the aid of automated deliverysystems, or robotics, and thus can provide an efficient alternative tocostly robotic delivery systems for laboratories using manualprocessing. Thus, the well plates, at systems, kits, and methods provideversatility in choice such that high throughput screening can be done ina cost effective manner, wherein the efficacy of the gene silencing usedin the screening can be tested along with the test siRNA.

The following terminology is defined herein to clarify the terms used indescribing embodiments of the present invention and is not intended tobe limiting. As such, the following terminology is provided tosupplement the understanding of such terms by one of ordinary skill inthe relevant art.

As used herein, the term “2′ modification” is meant to refer to achemical modification of a nucleotide that occurs at the second positionatom. As such, the 2′ modification can include the conjugation of achemical modification group to the 2′ carbon of the ribose ring of anucleotide, or a nucleotide within an oligonucleotide or polynucleotide.Thus, a 2′ modification occurs at the 2′ position atom of a nucleotide.Examples of a 2′ modification can include a 2′-O-aliphatic, 2′-O-alkyl,2′-O-methyl, 2′-O-ethyl, 2′-O-propyl, 2′-O-isopropyl, 2′-O-butyl,2′-O-isobutyl, 2′-O-ethyl-O-methyl (i.e., —CH₂CH₂OCH₃), 2′-O-ethyl-OH(i.e., —OCH₂CH₂OH), 2′-orthoester, 2′-ACE group orthoester, 2′-halogen,and the like.

As used herein, the term “antisense strand” is meant to refer to apolynucleotide or region of a polynucleotide that is at leastsubstantially (e.g., 80% or more) or 100% complementary to a targetnucleic acid of interest. Also, the antisense strand of a dsRNA iscomplementary to its sense strand. An antisense strand may be comprisedof a polynucleotide region that is RNA, DNA, or chimeric RNA/DNA.Additionally, any nucleotide within an antisense strand can be modifiedby including substituents coupled thereto, such as in a 2′ modification.The antisense strand can be modified with a diverse group of smallmolecules and/or conjugates. For example, an antisense strand may becomplementary, in whole or in part, to a molecule of messenger RNA(“mRNA” ), an RNA sequence that is not mRNA (e.g., tRNA, rRNA, and thelike), or a sequence of DNA that is either coding or non-coding. Theantisense strand includes the antisense region of polynucleotides thatare formed from two separate strands, as well as unimolecular siRNAsthat are capable of forming hairpin structures with complementary basepairs. The terms “antisense strand” and “antisense region” are intendedto be equivalent and are used interchangeably.

As used herein, the terms “complementary” and “complementarity” aremeant to refer to the ability of polynucleotides to form base pairs withone another. Base pairs are typically formed by hydrogen bonds betweennucleotide units in anti-parallel polynucleotide strands. Complementarypolynucleotide strands can base pair in the Watson-Crick manner (e.g., Ato T, A to U, C to G), or in any other manner that allows for theformation of duplexes. As persons skilled in the art are aware, whenusing RNA as opposed to DNA, uracil rather than thymine is the base thatis considered to be complementary to adenosine.

Perfect complementarity or 100% complementarity refers to the situationin which each nucleotide unit of one polynucleotide strand can hydrogenbond with a nucleotide unit of an anti-parallel polynucleotide strand.Less than perfect complementarity refers to the situation in which some,but not all, nucleotide units of two strands can hydrogen bond with eachother. For example, for two 20-mers, if only two base pairs on eachstrand can hydrogen bond with each other, the polynucleotide strandsexhibit 10% complementarity. In the same example, if 18 base pairs oneach strand can hydrogen bond with each other, the polynucleotidestrands exhibit 90% complementarity. “Substantial complementarity”refers to polynucleotide strands exhibiting 79% or greatercomplementarity, excluding regions of the polynucleotide strands, suchas overhangs, that are selected so as to be non-complementary.Accordingly, complementarity does not consider overhangs that areselected so as not to be similar or complementary to the nucleotides onthe anti-parallel strand.

As used herein, the term “conjugate” is meant to refer to a molecule,large molecule, or macromolecular structure that is coupled with eitherthe sense strand or antisense strand of an siRNA. That is, the moietycoupled to the siRNA is considered the conjugate. For clarity purposes,the siRNA can include a conjugate that is coupled thereto by a covalentbond, ionic interaction, and like couplings. Usually, a conjugate iscoupled with an siRNA in order to impart a functionality other thanincreasing the stabilization or targeting specificity. For examples,some conjugates, such as cholesterol, can be used to enhance the abilityof the siRNA to enter a cell. Other conjugates can be labels that can beused to detect transfection or the presence of the siRNA in the cell.Usually, the conjugate is coupled to the siRNA through a linker group.

As used herein, the term “control siRNA” is meant to refer to a type ofsiRNA that is well characterized, and can be used to test the efficacyof gene silencing that can be obtained from an RTF protocol. As such, acontrol siRNA can be used alone or with test siRNA and can provideresults that can be compared to known and established results that havebeen standardized for that control siRNA. A control siRNA can becharacterized as a positive control, negative control, and/or atransfection control. Control siRNA can be used to determine theefficacy of gene silencing that is being studied with a test siRNA.Control siRNA can be distinguished from test siRNA in that the controlsiRNA are well known and produce reproducable results, and are used as acontrol in a study that tests the ability of test siRNA to perform agene silencing function, whereas test siRNA can have known or novelsequences and are usually tested for functionality against a targetgene. Control siRNA are described in more detail below.

As used herein, the terms “dried” or “dry” as used in connection withgene silencing compositions is meant to refer to a composition that isnot fluidic and does not flow. However, this does not exclude smallamounts of water or other solvents, and includes amounts of waterremaining in an RNA preparation that has equilibrated at standard orambient conditions, for example, at one atmosphere of pressure, roomtemperature, and ambient humidity, such that the preparation is not in asubstantially liquid form but instead is “dried” in the well. Forexample, an siRNA preparation is “dried” or substantially “dry” if, atabout one atmosphere pressure, at about 20 to 40° C., and at about 50 toabout 95% humidity, the preparation is equilibrated and, when the wellplate is inverted or tilted to, for example, 90° from horizontal, theRNA preparation does not displace or flow within the well. This is incomparison to a liquid preparation which would flow or run when tilted.In various embodiments, methods for using the dry gene silencingcomposition in order to perform a transfection can include solubilizingor suspending the dried preparation in a suitable aqueous medium to forma mixture. Additionally, the suitable aqueous medium can include apolynucleotide carrier capable of facilitating introduction of the siRNAinto a cell, and exposing the mixture to one or more cells to achievetransfection.

As used herein, the term “duplex region” is meant to refer to the regionin two complementary or substantially complementary polynucleotides thatform base pairs with one another, either by Watson-Crick base pairing orany other manner that allows for a stabilized duplex between thepolynucleotide strands. For example, a polynucleotide strand having 21nucleotide units can base pair with another polynucleotide of 21nucleotide units, yet only 19 bases on each strand are complementarysuch that the “duplex region” has 19 base pairs. The remaining basesmay, for example, exist as 5′ and/or 3′ overhangs. Further, within theduplex region, 100% complementarity is not required, and substantialcomplementarity is allowable within a duplex region. Substantialcomplementarity refers to 79% or greater complementarity and can resultfrom mismatches and/or bulges. For example, a single mismatch in aduplex region consisting of 19 base pairs results in 94.7%complementarity, rendering the duplex region substantiallycomplementary.

As used herein, the term “functionality” is meant to refer to the levelof gene specific silencing induced by an siRNA. In general,functionality is expressed in terms of percentages of gene silencing.Thus, 90% silencing of a gene (e.g., F90) refers to situations in whichonly 10% of the normal levels of gene expression are observed.Similarly, 80% silencing of a gene (e.g., F80) refers to situations inwhich only 20% of the normal levels of gene expression are observed.

As used herein, the term “gene silencing” is meant to refer to a processby which the expression of a specific gene product is inhibited by beinglessened, attenuated, and/or terminated. Gene silencing can take placeby a variety of pathways. In one instance, gene silencing can refer to adecrease in gene product expression that results from the RNAi pathway,wherein an siRNA acts in concert with host proteins (e.g., RISC) todegrade mRNA in a sequence-dependent manner. Alternatively, genesilencing can refer to a decrease in gene product expression thatresults from siRNA mediated translation inhibition. In still anotheralternative, gene silencing can refer to a decrease in gene productexpression that results from siRNA mediated transcription inhibition.The level of gene silencing can be measured by a variety of methods,which can include measurement of transcript levels by Northern BlotAnalysis, B-DNA techniques, transcription-sensitive reporter constructs,expression profiling (e.g., DNA chips), and related technologies andassays. Alternatively, the level of gene silencing can be measured byassessing the level of the protein encoded by a specific gene that istranslated from the corresponding mRNA. This can be accomplished byperforming a number of studies including Western Blot analysis,measuring the levels of expression of a reporter protein, such ascalorimetric or fluorescent properties (e.g., GFP), enzymatic activity(e.g., alkaline phosphatases), or other well known analyticalprocedures.

As used herein, the term “nucleotide” is meant to refer to aribonucleotide, a deoxyribonucleotide, or modified form thereof, as wellas an analog thereof. Nucleotides include species that comprise purines,e.g., adenine, hypoxanthine, guanine, and their derivatives and analogs,as well as pyrimidines, e.g., cytosine, uracil, thymine, and theirderivatives and analogs. Nucleotide analogs include nucleotides havingmodifications in the chemical structure of the base, sugar and/orphosphate, including, but not limited to, 5′-position pyrimidinemodifications, 8′-position purine modifications, modifications atcytosine exocyclic amines, and 2′-position sugar modifications (e.g., 2′modifications). Such modifications include sugar-modifiedribonucleotides in which the 2′-OH is replaced by a group such as an H,OR, R, halo, SH, SR, NH₂, NHR, NR₂, or CN, wherein R is an alkyl oraliphatic moiety. Nucleotides are well known in the art. Also, referenceto a first nucleotide or nucleotide at a first position refers to thenucleotide at the 5′-most position of a duplex region, and the secondnucleotide is the next nucleotide toward the 3′ end. In instances theduplex region extends to the end of the siRNA, the 5′ terminalnucleotide can be the first by nucleotide.

As used herein, the term “polynucleotide” is meant to refer to polymersof nucleotides linked together through internucleotide linkages. Also, apolynucleotide includes DNA, RNA, DNA/RNA, hybrids includingpolynucleotide chains of regularly and/or irregularly alternatingdeoxyribosyl moieties and ribosyl moieties (i.e., wherein alternatenucleotide units have an —OH, then and —H, then an —OH, then an —H, andso on at the 2′ position of a sugar moiety), and modifications of thesekinds of polynucleotides. Also, polynucleotides include nucleotides withvarious modifications or having attachments of various entities ormoieties to the nucleotide units at any position.

As used herein, the terms “rational design” and “rationally designed”are meant to refer to the selection or design of one or more siRNA(s)for use in a gene silencing application based upon one or more criteriathat are independent of the target sequence. As such, rationallydesigned siRNA are selected to specifically interact with and inhibitpolypeptide translation from a selected mRNA. Thus, for any one targetmRNA there may be hundreds of potential siRNA having from 18 to 31 basepairs that are 100% complementary to the target mRNA. In part, this isbecause a single mRNA may have multiple sequences that can bespecifically targeted by the siRNA. However, it is likely that not allof the siRNA will have equal functionality. Through empirical studies, anumber of other factors including the presence or absence of certainnitrogenous bases at certain positions, the relative GC content, and thelike, can affect the functionality of particular siRNA. Additionalinformation regarding rationally designed siRNA can be found in commonlyowned U.S. patent application Ser. No. 10/714,333, filed on Nov. 14,2003, related PCT application PCT/US03/36787, published on Jun. 3, 2004as WO 2004/045543 A2, U.S. patent application Ser. No. 10/940,892, filedon Sep. 14, 2004, published as U.S. Patent Application Publication2005/0255487, related PCT application PCT/US 04/14885, filed on May 12,2004, and U.S. Patent Application Publication 2005/0246794, which areall incorporated herein by reference.

As used herein, the term “reverse transfection” and abbreviation “RTF”are each meant to refer to a process for introducing nucleic acid, suchas an siRNA, into a cell. Such an introduction of an siRNA into a cellcan be accomplished by combining the nucleic acid and cell in a well,wherein the cell has not yet been previously adhered or maintained onthe growth surface. The reverse transfection proceeds by contacting thenucleic acid onto a cellular surface in a manner such that the nucleicacid can enter into the cell. Usually, the siRNA is complexed with alipid or other polynucleotide carrier prior to being contacted to thecells. Reverse transfection differs from forward transfection becausethe cells have not been seeded and maintained on the cellular growthsurface of a well or other container before addition of the siRNA.

As used herein, the term “sense strand” is meant to refer to apolynucleotide or region that has the same nucleotide sequence, in wholeor in part, as a target nucleic acid such as a messenger RNA or asequence of DNA. The term “sense strand” includes the sense region of apolynucleotide that forms a duplex with an antisense region of anotherpolynucleotide. Also, a sense strand can be a first polynucleotidesequence that forms a duplex with a second polynucleotide sequence onthe same unimolecular polynucleotide that includes both the first andsecond polynucleotide sequences. As such, a sense strand can include oneportion of a unimolecular siRNA that is capable of forming hairpinstructure, such as an shRNA. When a sequence is provided, by convention,unless otherwise indicated, it is the sense strand or region, and thepresence of the complementary antisense strand or region is implicit.The phrases “sense strand” and “sense region” are intended to beequivalent and are used interchangeably.

As used herein, the term “siRNA” is meant to refer to a small inhibitoryRNA duplex that induces gene silencing by operating within the RNAinterference (“RNAi”) pathway. These siRNA are dsRNA that can vary inlength, and can contain varying degrees of complementarity between theantisense and sense strands, and between the antisense strand and thetarget sequence. Each siRNA can include between 17 and 31 base pairs,more preferably between 18 and 26 base pairs, and most preferably 19 and21 base pairs. Some, but not all, siRNA have unpaired overhangingnucleotides on the 5′ and/or 3′ end of the sense strand and/or theantisense strand. Additionally, the term “siRNA” includes duplexes oftwo separate strands, as well as single strands that can form hairpinstructures comprising a duplex region, which may be referred to as shorthairpin RNA (“shRNA”).

As used herein, the terms “siRNA pool,” “pool,” “pool of siRNAs,” and“pool reagents” are meant to refer to two or more siRNA, typically foursiRNA, directed against a single target gene, mRNA, and/or translationof a protein. The siRNA of the pool reagent can be rationally designedby being selected according to non-target specific criteria as describedherein and in the incorporated references. For example, two nanomoles ofeach pool reagent can be sufficient for transfecting cells in about 200wells of multiple 96-well plates, using 100 nM siRNA concentration. Poolreagents can be plated as a pool (i.e., the two or more siRNA ofDharmacon's SMARTpool® Reagent in a single transfection well). Theindividual siRNAs that comprise the SMARTpool® Reagent can also beplated individually on the same plate as the SMARTpool® Reagent.

As used herein, the term “target” is used in a variety of differentforms throughout this document and is defined by the context in which itis used. The term “target gene” is meant to refer to the gene thatencodes the protein to be silenced by the siRNA, and encodes for theproduction of the target mRNA. The term “target mRNA” is meant to referto an mRNA against which a given siRNA is direct to silence thetranscription of the polypeptide product. The term “target sequence” and“target site” are meant to refer to a sequence within the mRNA, miRNA,or DNA coding or promoter region to which the sense strand of an siRNAexhibits varying degrees of homology and the antisense strand exhibitsvarying degrees of complementarity. The term “target polypeptide” or“target protein” is meant to refer to the gene product encoded by thetarget gene, target mRNA, and/or target sequence. The term “siRNAtarget” can refer to the gene, mRNA, or protein against which the siRNAis directed to for silencing. Similarly, “target silencing” can refer tothe state of silencing a gene, or the corresponding mRNA or protein.

As used herein, the term “transfection” is meant to refer to a processby which nucleic acids are introduced into a cell. The list of nucleicacids that can be transfected is large and includes, but is not limitedto, siRNA, shRNA, sense and/or anti-sense sequences, DNA, RNA, and thelike. There are multiple modes for transfecting nucleic acids into acell including, but not limited to, electroporation, calcium phosphatedelivery, DEAE-dextran delivery, lipid delivery, polymer delivery,molecular conjugate delivery (e.g., polylysine-DNA or -RNA conjugates,antibody-polypeptide conjugates, antibody-polymer conjugates,cholesterol conjugates, or peptide conjugates), microinjection, laser-or light-assisted microinjection, optoporation or photoporation withvisible and/or nonvisible wavelengths of electromagnetic radiation, andthe like. Transfections can be “forward transfections” whereby cells arefirst plated in wells and then treated with a nucleic acid or they canbe “reverse transfections” (RTF) whereby the nucleic acid is combinedwith the cells before or during being plated and/or attached to thebottom of the well. Any mode of transfecting cells, such as thosedescribed above, can be used with the present invention by inducing thenucleic acid to be introduced into a cell after the siRNA is solubilizedor suspended in the aqueous medium to implement reverse transfection.Details regarding a mode of reverse transfection are described in moredetail below

As used herein, the term “well plate” is meant to refer to a substratethat is divided into distinct regions that prevent migration from onedistinct region to another distinct region, wherein the distinct regionsare wells. For example, each well of a multi-well well plate may containa horizontal well floor that may be curved or flat, as well as havesidewalls. Additionally, well plates are well known in the art.

The use of units to define measurable quantities of material, such asconcentration, weight, and volume, are intended to be those that areroutinely employed by those of skill in the art. Additionally, the unitsare preferably interpreted to correspond with the metric system. Also,the use of “u,” as in “ug” or “uL” is meant to refer to “micro” asapplied to microgram and microliter, respectively.

Additionally, while the foregoing term definitions are intended tosupplement the knowledge of one of ordinary skill in the art, not everyterm within this document has been defined. As such, the undefined termsare intended to be construed with the knowledge of one of ordinary skillin the art and/or the plain meaning of the term. Additionally, theforegoing terms are not intended to be limited by the examples providedtherein, but are intended to be useful in understanding and practicingthe invention as described herein.

I. Reverse Transfection

Generally, the present invention provides well plates, systems, kits,and methods for testing and/or optimizing the efficacy of proceduresand/or conditions that implement reverse transfection (“RTF”) of siRNA.As such, the present invention can provide for plates, systems, kits,and methods that can be used to assess or test the efficacy of genesilencing. The present invention provides methods of determining orassessing the efficacy of gene silencing so that improvements in bothreverse transfection methodologies that pertain to siRNA and theefficiency of siRNA based gene silencing can be obtained. In part, thisis because the plates, systems, kits, and methods use control siRNA thatcan provide meaningful results pertaining to the accuracy of resultsobtained from a gene silencing protocol. Thus, the results obtained fromusing control siRNA can be used to validate or invalidate gene silencingresults, and lead to the improvement of siRNA RTF protocols and theconditions used therewith.

In one embodiment, the present invention includes a method of testingand/or optimizing the effectiveness of an siRNA RTF protocol and/orcondition for introducing siRNA into a cell to effect gene silencing.Such a method can include providing a well plate that includes a wellhaving a substantially dry control composition. The control compositioncan include an individual control siRNA or a a pool of control siRNAs.The control composition can include a control siRNA which can be used toprovide meaningful information regarding the effectiveness of the siRNARTF protocol and/or condition. Such meaningful information can providean indication of whether or not the RTF protocol and/or condition issufficient for introducing test siRNA into cells to effect genesilencing. Also, the meaningful information can provide an indication ofwhether or not any gene silencing data obtained from the siRNA RTFprotocol is valid and reliable, or whether the data inaccuratelyrepresents the effectiveness of a test siRNA to silence a target gene.Also, the control siRNA can provide an indication of whether any genesilencing is a function of cellular toxicity or non-specific genesilencing rather than that resulting from specific gene silencing.Moreover, the method of testing the effectiveness of an siRNA RTFprotocol and/or condition can be used as part of an optimizationprocedure so that the optimum gene silencing conditions can be selectedfor certain siRNA, cells, polynucleotide carriers, and the like.

The control siRNA can be present in the well of a plate as part of a drycontrol composition so that the test plates or optimization plates canbe prepared, sealed, stored, and/or shipped long before an RTF testingprotocol is performed. In part, this is because the dry controlcomposition can stably retain the control siRNA in a usable conditionwithin the well, and be resuspended or resolubilized with an aqueousmedium during the RTF testing protocol. Thus, a well plate having thecontrol composition can be manufactured and hermetically sealed in aninert environment within a sterile package, wherein the plate caninclude different wells with predefined types of control siRNA andoptionally test siRNA for specific gene targets. Such types of controlsiRNA intended to test the effectiveness of an siRNA RTF protocol aredescribed in more detail below.

In any event, the testing can be performed by adding an aqueous mediumto each well that contains a control composition so as to suspend orsolubilize the control siRNA into the aqueous medium. The aqueous mediumis allowed to solubilize or suspend the control siRNA for a sufficientduration so that most, if not all, of the control siRNA is solubilizedor suspended. Optionally, the aqueous medium or an additional solutionis comprised of a polynucleotide carrier. However, polynucleotidecarriers are not necessary in some embodiments.

After the control siRNA is adequately solubilized or suspended, cellsare added to the well under conditions that permit the control siRNA tobe introduced into the cell. The cells can be added in an amount ofabout 1×10³ to about 3.5×10⁴ cells per about 0.3 cm² to about 0.35 cm²of cell growth surface area. The conditions that promote the controlsiRNA entering the cell can be described by typical cell culturetechniques used for plating cells that are well known in the art, andfurther can be the conditions used in an siRNA RTF protocol to betested. That is, the cells can be added to the well that contains thecontrol siRNA in a manner similar to ordinary plating. Optionally, thecells are added to a well having a dry control composition so that theaqueous medium carrying the cells can solubilize or suspend the controlsiRNA. The well containing the control siRNA and cells can be incubatedfor a sufficient duration for gene silencing to occur, which istypically less than 72 hours, more preferably less than 48 hours, andmost preferably about 24 hours or less.

In one embodiment, the methods include transfecting the cells with thesiRNA. As such, any mode of transfection can be implemented in the RTFformat by adding the cells to the well having the siRNA. Accordingly,the cells can be transfected while suspended or while attaching to thewell floor. The modes of transfection can include those described aboveor others known or developed later.

In one embodiment, the RTF testing protocol can include adding thepolynucleotide carrier to the well so as to form a control complex,wherein the control complex is suspended or solubilized in the aqueousmedium. After the cells are added, the control complex can be contactedto the cell to induce endocytosis of the complex. As such, thepolynucleotide carrier can be added as part of the aqueous medium or inaddition thereto. Thus, the polynucleotide carrier can be presented inan aqueous medium and be either solubilized or suspended therein. Thepolynucleotide carrier can be a cationic lipid, polymer, lipopolymer,and the like.

After the cells are combined with the control siRNA, the well plate canbe maintained under conditions so that cell growth, cell division,transfection, and/or gene silencing occurs. Usually, the cells aremaintained in the presence of the control siRNA for about 6 to about 72hours before gene silencing is assessed, more preferably about 12 toabout 36 hours, and most preferably for about 24 to about 48 hours.However, it should be recognized that the cells are incubated with thecontrol siRNA for a time period sufficient for silencing a gene so thatthe amount corresponding gene product decreases. As such, the productionof a target polypeptide can be silenced by at least about 50%, morepreferably by at least about 70%, even more preferably by at least about80%, and most preferably by at least about 90%.

In instances where cells that grow in suspension are the target cell,such cells can be added to the wells at an appropriate cell density andplates can be spun under low gravity forces that are not detrimental tocell viability to bring the cells and lipids into close proximity on thebottom of the well.

In one embodiment, the control composition includes a positive controlsiRNA. The positive control siRNA can be characterized by being capableof silencing expression of a known gene. Also, the positive controlsiRNA can provide consistent, reproducible, and known results that canbe used as assess the gene silencing efficacy of an RTF protocol and/orcondition. For example, the positive control siRNA can silence at leastone of a MAP kinase gene, cyclophilin B gene, lamin A/C gene,glyceraldehyde-3-phosphate dehydrogenase gene or other well-known andestablished control genes that can be reproducibly silenced

In one embodiment, the control composition includes a transfectioncontrol siRNA. The transfection control siRNA can provide the ability toassess the level or percentage of cells that have been successfullytransfected with the transfection control siRNA. That is, a transfectioncontrol siRNA can be configured to identify whether or not it hassuccessfully entered into a cell. In one aspect, the transfectioncontrol siRNA includes a label that can be detected in a cell. Thisallows the cells to be analyzed to determine whether or not thetransfection control siRNA is present in the cell. Examples of labelsinclude colorimetric labels, chemiluminescent labels, fluorescentlabels, mass labels, and radioactive labels. The labels can beconsidered conjugates and can be coupled with siRNA as described forconjugates herein and in the incorporated references. This includes adirect coupling to the siRNA and coupling through a linker. The labelcan be coupled to a 5′ terminal nucleotide or 3′ terminal nucleotide onone of a sense strand or an antisense strand. Optionally, the label canbe attached to any nucleotide on the sense strand or antisense strand.However, it is preferable for the label to be fluorescent, and becoupled to the sense strand.

In one embodiment, the transfection control siRNA can be toxic to cells.This can include siRNA that include at least one toxic motif. That is,the siRNA includes a polynucleotide sequence that can lead to cellulartoxicity and/or cell death. Examples of such toxic motifs can includesiRNA that include a polynucleotide sequence including an AAA motif, UUUmotif, GCCA motif, or UGGC motif. However, other toxic siRNA can beemployed as transfection control siRNA.

In one embodiment, the control composition includes negative controlsiRNA. The negative control siRNA can be configured to be non-functionalsiRNA. That is, the siRNA can include a polynucleotide sequence thatdoes not function in the RNAi pathway. For example, the negative controlsiRNA can have a polynucleotide sequence that does not target any knownhuman gene, animal gene, or gene within the cell being transfected andstudied. Also, a non-functional siRNA can include a 17 base pair duplexcontaining a sense strand with 2′ modifications at the first and secondnucleotide, and an antisense strand with 2′ modifications at the firstand second nucleotide. Alternatively, the non-functional siRNA caninclude a 19 base pair duplex having 5′ deoxy nucleotides on the 5′terminal nucleotides of the sense and/or antisense strands.

In one embodiment, the negative control siRNA can be configured toinhibit RISC uptake and processing. This can include a chemicallymodified siRNA that inhibits being taken in or processed by RISC.

In one embodiment, the cells transfected with the control siRNA duringthe RTF testing protocol can be assessed for cell viability, controlgene silencing, control polypeptide production, control mRNA amount,presence of the control siRNA, non-functionality of the control siRNA,and the like. The cell viability studies can be performed in the wellplate in accordance with well known procedures. Additionally, thecontrol gene silencing can also be assessed with the contents in thewell by various techniques well known in the art to assess the presenceor absence of target proteins. When the control siRNA includes a label,it can be detected in the cell. Alternatively, the amount of genesilencing can be assessed by removing the contents from the well by wellknown assays. In various embodiments, the well is designed to becompatible with optical detection systems such as, for example, UV,luminescence, fluorescence, or light scattering detection systems, whichcan be favorable for transfection control siRNA having fluorescentlabels. In embodiments compatible with optical detection systems, thewalls of the well can be made opaque, or rendered such that lightscattering that can interfere with optical detection is reduced orminimized.

In one embodiment, the results of the RTF protocol to induce genesilencing can be detected or monitored using systems for performing highcontent screening (“HCS”) or high throughput screening (“HTS⇄). An HCSanalysis can be used to measure specific translocation and morphologychanges, receptor trafficking, cytotoxicity, cell mobility, cellspreading, and the like. HCS studies can be performed on an ArrayScan®HCS Reader, or a KineticScan® HCS Reader (Cellomics, Inc.) Additionalinformation on HCS can be found in U.S. Pat. Nos. 6,902,883, 6,875,578,6,759,206, 6,716,588, 6,671,624, 6,620,591, 6,573,039, 6,416,959,5,989,835, wherein each is incorporated herein by reference. HTSanalyses can be performed using a variety of available readers,typically of the fluorescence from each well as a single measurement.

In one embodiment, the invention includes a well plate configured forhaving the contents of a well transferred to a location, device, orsystem wherein detection of the results of an siRNA RTF testing protocolis carried out. As such, wet transfer detection systems can be employedthat include systems wherein cells are transferred from wells to asubstrate such as nitrocellulose. Following the transfer of the wellcontents to the substrate a detection protocol can be implemented. Anexample of such a well plate transfer system can include nitrocellulose,wherein the well contents can be treated such that cell membranes arepermeabilized or disrupted so as to gain access to intracellularcontents. The transfer of the well contents to the nitrocellulose can beachieved by any suitable method including gravity or use of a vacuummanifold. The nitrocellulose containing the well contents can then befurther subjected to a detection protocol that uses antibody-baseddetection systems and the like to detect the presence or level of one ormore contents of the cells that comprise a particular well.

II. Optimizing siRNA RTF

Due to the unique and highly sensitive nature of the RNAi pathway,methodologies particularly useful for introducing siRNA into cells havebeen developed. Recently developed protocols for implementing siRNA RTFwere modified by augmenting such protocols with recently developed siRNAtechnologies based on rationale design, siRNA stabilization, siRNAtargeting specificity, and pooling siRNAs. However, the effectiveness ofnew siRNA RTF protocols can still be compromised by a lack ofoptimization, errors, or improper conditions. Thus, methods for testingand optimizing the efficacy of gene silencing with siRNA RTF formats arepresented herein.

It has recently been discovered that there are many diverse responseswhen different cell numbers, different lipids, and different lipid tosiRNA ratios are used in an RTF format in comparison with those that arerecommended for forward transfection protocols. Accordingly, controlsiRNA can be used in RTF testing protocols to assess the conditions inwhich a test siRNA is employed.

The number of cells per well, which is referred to as the cell density,is an important parameter of successful siRNA pool RTF. It has beenfound that siRNA pool RTF protocols can have more favorable results withlower cell densities compared to RTF protocols using DNA. For example,96-well plates can include cell densities of about 1,000-35,000 cellsper well, more preferably about 1,250-30,000 cells per well, even morepreferred are cell densities of about 1,500-20,000 cells per well, stillmore preferably about 1,750-15,000 cells per well, and most preferableare cell densities of about 3,000-10,000 cells per well. Also, thenumber of cells per well can be extrapolated to wells having differentcell culture areas. One possible equation for calculating theappropriate number of cells that are placed in a given well is based ona 96-well plate having a cell culture area of about 0.3 cm² to about0.35 cm², wherein well #2 is the 96-well plate, and is described asfollows:${{cells}\quad{in}\quad{well}\quad{\# 1}} = {\left( \frac{{area}\quad{of}\quad{well}\quad{\# 1}}{{area}\quad{of}\quad{well}\quad{\# 2}} \right) \times {cells}\quad{in}\quad{well}\quad{\# 2}}$

Additionally, siRNA RTF testing protocols can be used to determinewhether a particular polynucleotide carrier, such as a lipid, can beuseful in a particular siRNA RTF condition. The polynucleotide carriercan be tested over a wide range of carrier concentrations by using arobust and easily-transfected cell line (e.g., HeLa) with control siRNAover commonly used ranges of cell density and siRNA concentrations.Accordingly, cell viability and the function of the control siRNA can beassayed with the foregoing concentration gradients. Thus, optimizationstudies can be performed with concentration gradients in order todetermine which polynucleotide carriers can produce highly efficienttransfection without inducing unfavorable cell toxicity.

In one embodiment, the present invention is directed to optimization ofsiRNA RTF protocols for implementing gene silencing through the RNAipathway. As such, optimization of siRNA RTF can include any of thefollowing: (1) selecting the type of plate; (2) selecting an appropriatesolution to solubilize or suspend the siRNA for being deposited anddried in a well; (3) selecting at least one control siRNA or a pool ofcontrol siRNAs, which can be a transfection control, positive control,or a negative control; (4) identifying any modifications or conjugatesthat can be applied to the individual siRNA in order to enhance siRNAstability and/or specificity; (5) applying and drying the siRNA on asolid surface so that it can be solubilized or suspended in anappropriate aqueous medium; (6) selecting an appropriate mode oftransfection; (7) selecting a polynucleotide carrier for siRNA such as alipid; (8) solubilizing or suspending an siRNA; (9) complexing the siRNAwith the polynucleotide carrier to form an siRNA-carrier complex; and(10) combining the siRNA-carrier complex with the cell type or types ofchoice. Thus, optimizing siRNA RTF protocols can result in a dramaticimprovement over previous forward and reverse transfection procedures.

In one embodiment, the present invention can include siRNA RTF testprotocols to implement along with the foregoing optimizations, which caninclude any of the following: (a) applying at least one control siRNA totwo or more wells of a multi-well plate; (b) drying the control siRNA onthe floor of each well; (c) adding, an aqueous medium such as a media orbuffer to the control siRNA in each well in order to solubilize orsuspend the control siRNA, and optionally the solution includes apolynucleotide carrier so that a control complex can form; (d) adding anappropriate number of cells to each well in which the control siRNA isalready in solution alone or as an control complex; and (e) after cellshave been added, maintaining the plate under conditions in whichtransfection of the cells by the control siRNA can occur. Followingtransfection, the cells are subjected to conditions, such as liquidmedia, temperature, gas partial pressures, and the like, in which cellgrowth and/or cell division will occur and gene silencing may occur.These conditions can be, but not necessarily, the same as the conditionsunder which transfection occurs, and are well known in the art.

III. Well Plates

In one embodiment, the present invention includes the use of controlsolutions dried in the bottom of a well in a well plate. The well platesused in connection with the present invention are preferably formattedand distinct well arrays (e.g., a 48, 96, 384, or 1536 well plate) thatcan be purchased from any number of commercial sources of cell cultureplates and other cell culture surface-containing devices.

A well plate can be made of glass, polystyrene, other polymeric materialor any equivalent materials, and can form a rounded or generally flathorizontal bottom having various generally planar shapes. Additionally,wells having substantially flat floors can provide uniform cell spacingand monolayer formation and are preferred. Additionally, it can bepreferable for each plate to have between 32 and 2000 wells, and morepreferably having 1536 wells, 384 wells, or 96 wells; however, a platehaving any number of wells can be used. Also, it can be preferable forthe wells to have a volume that varies between about 5 to about 200microliters (“uL”), and the total culture area, which is represented bythe well floor, to range between about 0.02 cm² and about 0.35 cm².Additionally, the wells may not be modified by any chemical coating, orthey can be coated with poly-L-lysine (“PLL”), laminin, collagen, orequivalent substances that improve the adherence of cells. Additionaldescriptions of well plates can be reviewed in the cross-referencedapplication having Attorney Docket No. 16542.1.1, entitled APPARATUS ANDSYSTEM HAVING DRY GENE SILENCING COMPOSITIONS, with Barbara Robertson,Ph.D., et al. as inventors, which is incorporated herein by reference.

Additionally, any of the plates can be included in systems or kits inaccordance with the present invention. Such kits can include the plateshaving control compositions and can be distributed with siRNAsolubilizing or suspending solutions, polynucleotide carriers, carriersolutions, reagents, cell media, and the like.

IV. Reverse Transfection Testing Plates

In one embodiment of the present invention, a well plate in accordancewith the foregoing can be configured as a reverse transfection testingplate (“RTF testing plate”). Accordingly, the RTF testing plate caninclude a control composition in one or more wells. The controlcomposition includes at least a first control siRNA that can provide anindication of the efficacy of gene silencing. Optionally, the controlcomposition can have a pool of control siRNAs. Well plates can be RTFtesting plates by having a control siRNA-containing solution applied toat least one well, which is then dried in a manner that removes thesolution and leaves a dried control composition.

In some instances the control siRNA can be solubilized in one of severaltypes of solutions prior to applying, depositing, and/or spotting thecontrol siRNA solution onto the well floor, and drying the material onthe plate. The control siRNA can be dissolved in distilled water thathas been treated by one of any number of art-recognized techniques toeliminate contamination by RNases such as by ultrafiltration.Alternatively, the control siRNA may be dissolved in one of severalphysiologically compatible, RNase-free buffers, including but notlimited to phosphate buffer, Hanks BSS, Earl's BSS, or physiologicalsaline. These solutions may contain one or more additional reagents thatenhance the stability of the control siRNA (e.g., RNase inhibitors) oralter the viscosity of the solution to enhance spotting or dryingefficiency (e.g., sucrose) without changing the properties of thecontrol siRNA or injuring the cells that are added at subsequent stagesin the RTF testing protocol.

In still other cases, the control siRNA may be solubilized in a solutionor medium that will enhance spotting, drying, or sticking to the plateof choice. Optionally, volatile solvents can be used that are compatiblewith control siRNA. One example includes the use of alcohols, such asethanol, which can be mixed with water in order to form a volatilesolvent that can be readily dried and leave a dry control composition onthe well floor. In some instances the solution of control siRNA does notcontain lipids that are easily oxidized over the course of time or canbe toxic to cells. In other instances the control siRNA is pre-complexedwith a polynucleotide carrier in a solution before being deposited anddried to the well floor.

Accordingly, a predefined amount of control siRNA can be administered tothe well so that when it is dried and then resuspended, a known amountor concentration of control siRNA is available for testing genesilencing. The volume of solutions that are deposited on the bottom ofeach well can depend upon the concentration of the stock solution,functionality of the control siRNA, and desired amount or concentrationof control siRNA available for testing gene silencing. For example, theconcentration of siRNA during transfection can range from picomolar(e.g., 300-900 pM) for highly functional siRNA (e.g., silence >90% oftarget expression at 50-100 nM), to nanomolar (e.g., 100 nM) for siRNAof intermediate functionality (e.g., 70-90% silencing of targetexpression at 50-100 nM), and to micromolar (e.g., 1 uM) for lowfunctionality. For example, for a 96-well plate, deposition of 5-50 uLof a 1 uM siRNA-containing solution is sufficient to generate anacceptable concentration of control siRNA for RTF testing protocols. Forsmaller or larger sized wells, volumes and amounts of control siRNAwould be adjusted to compensate for the final concentration oflipid-media/buffer and media that can be accommodated in each well.

In one embodiment, the total amount of siRNA in the control compositioncan be present in an amount for transfecting cells in only the well inwhich it is contained. As such, the total concentration of control siRNAcan be less than about 100 nM when solubilized or suspended in theaqueous medium during RTF, more preferably less than about 50 nM, evenmore preferably the total concentration of siRNA can be less than about25 nM, and most preferably less than about 10 nM when solubilized orsuspended in the aqueous medium during RTF. In another option, the totalconcentration of control siRNA can be less than about 1 nM whensolubilized or suspended in the aqueous medium during RTF. For example,the amount of control siRNA in a 96-well plate can be from 0.1 picomoles(“pm”) to about 100 pm, more preferably about 1 pm to about 75 pm, andmost preferably about 10 pm to about 62.5 pm per well, wherecorresponding amounts of control siRNA can be calculated for plateshaving other numbers of wells.

Additionally, the amount of control siRNA added to each well can besufficient for use in a single RTF testing protocol within that well.That is, the control siRNA in the control composition can be present inan amount to only be used with the cells added to the well. As such, theamount of control siRNA dried in the well can be insufficient forperforming two RTF protocols in two different wells. This is because theamount of control siRNA provided in the control composition isconfigured for a single RTF testing protocol in order to produce optimalresults. Also, this eliminates the need to make a stock siRNA solutionthat is transferred into multiple wells, thereby reducing the complexityof the RTF testing protocol and increasing efficacy.

The control siRNA-containing solutions can be deposited into wells usingvarious well known techniques in the art for depositing liquids intowells of well plates, which can include manual and automated processes.Various methods can be used to dry the control siRNA-containing solutioninto a control composition. In one embodiment, the plates are allowed todry at room temperature in a sterile setting which allows the depositionsolution to evaporate leaving behind the control siRNA and any otherconditioning compounds, such as salts, sugars, and the like. Driedplates are preferably vacuum-sealed or sealed in the presence of inertgases within a sterile container, and stored at temperatures rangingfrom −80° C. to 37° C. for extended periods of time without loss ofsilencing functionality. Thus, the plates having the dry controlcompositions in at least one well can be stored at room temperature andshipped via traditional routes and still maintain the integrity andfunctionality of the control siRNA.

In one embodiment, the plate can further include a blank well devoid orsubstantially devoid of siRNA. That is, the blank well does not haveenough siRNA to provide meaningful gene silencing. The blank well can beused in concert with control siRNA to show the effects of the genesilencing condition in the absence of any siRNA. This can be especiallybeneficial when assessing the toxicity of test or control siRNA, ormeasuring the amount of a particular mRNA or protein present in a cell.Also, this can provide data regarding the effect of the polynucleotidecarrier on the cell in the absence of siRNA.

In one embodiment, the plate can further comprise at least one test wellthat has a substantially dry gene silencing composition. The genesilencing composition can have at least a first test siRNA whichsilences a test target gene. Additionally, the gene silencingcomposition can be configured such that the test siRNA is capable ofbeing solubilized or suspended in an aqueous medium in an amountsufficient for transfecting cells in the well. The gene silencingcomposition can be characterized by at least one of the following: (a)the test siRNA can be unmodified; (b) the test siRNA can have astabilizing modification;(c) the test siRNA can have a modification tolimit off-targeting; (d) the test siRNA can have a conjugate; or (e) thetest siRNA can have a hairpin structure.

V. Control siRNA

In one embodiment, the dry control compositions include at least a firstcontrol siRNA which can provide an indication of the efficacy of genesilencing. Optionally, the dry control composition can include a pool ofcontrol siRNAs. The control composition is configured such that thecontrol siRNA is capable of being solubilized or suspended in an aqueousmedium in an amount sufficient for transfecting cells in the well.Optionally, the total amount of control siRNA in the well is sufficientfor implementing reverse transfection only for that well. Additionally,it is optional for the control siRNA to have at least one of amodification or a conjugate. Also, the control siRNA can be rationallydesigned to target a control gene. Furthermore, the control compositioncan include a pool of control siRNAs. Examples of control siRNA includetransfection control siRNA, positive control siRNA, and negative controlsiRNA.

The ability to use control siRNA in a control well to test the efficacyof gene silencing in other test wells is based on the consistencybetween RTF protocols and conditions. In part, consistency betweenconditions of a control well and a test well may enable the results ofthe control well to provide meaningful results with regard to the testwell. Consistency between the control well and test well can includeconsistency in cell type, cell seeding number, cell density, mode oftransfection, polynucleotide carrier type, polynucleotide carrierconcentration, siRNA concentration, cell culture media, anyenvironmental conditions to which the both wells are subjected, and thelike. In the instance in which a number of factors are different betweenthe control well and the test well, the results obtained from thecontrol well may not be indicative of the gene silencing efficacy of thetest siRNA. Thus, it can be beneficial for the control well and testwell to be on the same plate, or run in concert with conditions andprotocols that are as similar as possible.

Often, control siRNA are utilized to determine whether or not theprotocol or conditions associated with RTF protocol can inducenon-specific gene silencing or, in some instances, a unique phenotype.The results obtained from using control siRNA can then be compared tothe test data obtained from test siRNA, wherein the test siRNA are thesiRNA that are being studied in the gene silencing experiments.Additionally, the control siRNA can be characterized by the following:(a) the control siRNA can be unmodified; (b) the control siRNA can havea stabilizing modification; (c) the control siRNA can have amodification to limit off-targeting; (d) the control siRNA can have aconjugate; or (e) the control siRNA can have a hairpin structure.

A. Transfection Control siRNA

Transfection control siRNA are configured to enable the level orpercentage of cells that have been transfected during an RTF testingprotocol to be identified and/or quantified. This includes control siRNAthat can be used to monitor the efficiency of an RTF procedure and/orcondition. The level of transfection or percentage of cells that havebeen transfected can be measured by a variety of methods, which includeidentifying the presence of transfected control siRNA in a cell ormeasuring the effects of the control siRNA on the cell. When the effectsof control siRNA are measured, the effects can be direct or indirect,but are usually well known and reproducible effects that arespecifically caused by the control siRNA. In any event, transfectioncontrol siRNA are used to monitor the efficacy of an RTF protocol and/orcondition by comparing the resulting effects in the control well withestablished and reproducible effects that correspond with the specifictransfection control siRNA. This provides an indication of thetransfection and/or gene silencing efficiency.

In the instance where the presence of the transfection control siRNA ina transfected cell is identified or quantified during an RTF testingprotocol, the control siRNA can be a type that is detected withoutregard to any functionality. That is, the control siRNA can beconfigured to be detected in a cell without measuring any effect thatmay have been induced. Such transfection control siRNA can include alabel that can be directly measured by techniques well known in the art.Examples of labels can include calorimetric labels, fluorescent labels,luminescent labels, chemiluminescent labels, enzymatic labels, masslabels, radioactive labels, and the like. Thus, the presence of thecontrol siRNA in a cell can be measured within the contents of the cellbeing retained within the well, or the contents can be removed foranalysis. Any untransfected siRNA can be removed from the cell cultureby well known washing protocols.

In one embodiment, the transfection control siRNA includes acolorimetric label. A colorimetric label can be a chromophore that isdetectible by measuring and analyzing the absorbance or transmittedcolor spectrum or single wavelength of a sample. As such a chromophorecan be coupled to the siRNA so that the transfection can be detected ormeasured by the color spectrum or single wavelength that is transmittedin response to incident light or measuring the absorbance of the sample.This can also be compared to the color spectrum that can be obtainedfrom cells in a blank well. Alternatively, a colorimetric label can bean enzyme that reacts with a specific substrate to generate achromophore product. The label can be measured and/or quantified bymeasuring the amount of light absorbed or transmitted by the product ata specific wavelength or spectrum. For example, enzymes that can be usedas labels include alkaline phosphatase with para-nitrophenyl phosphateas the substrate, horseradish peroxidase with hydrogen peroxide/coupleras the substrate, β-galactosidase with O-nitrophenylgalactoside assubstrate and the like.

In one embodiment, the transfection control siRNA includes a fluorescentlabel. The fluorescent label can be used in order to photometricallymonitor the delivery of the control siRNA into a cell. Preferably, thefluorescent label is a rhodamine or a fluorescing however, otherfluorescent molecules that can be coupled with an siRNA can be used.Specific examples of fluorescent labels include Cy3™, Cy5™ (Amersham),other cyanine derivatives, FITC, one of the ALEXA™ or BODIPY™ dyes(Molecular Probes, Eugene, Oreg.), a dabsyl moiety, and the like. It isalso possible to use fluorescent microparticles, such as inorganicfluorescent particles as long as the particle has a size that does notaffect transfection efficiencies. The labels may be used to visualizethe distribution of the labeled siRNA within a transfected cell. Inaddition, the label can be used to distinguish between transfected cellsfrom non-transfected cells. As such, a population of cells can betransfected with the labeled siRNA and sorted by FACS. Moreover, thefluorescent labels can be particularly well suited for HCS and HTCanalytical techniques. For example, cells that have been transfected canbe identified, and then be further examined using HCS analysis.

In one embodiment, the label can be a luminescent moiety other than afluorescent label. Such luminescent moieties can include phosphorescentmicroparticles that can be coupled to siRNA. Preferably, the size of thephosphorescent microparticle does not affect transfection.Alternatively, the luminescent label can be a chemiluminescent moiety. Achemiluminescent label can produce light by a chemical orelectrochemical reaction. Chemiluminescence usually involves theoxidation of an organic compound, such as luminol or acridinium esters,by an oxidant like hydrogen peroxide or hypochlorite. Also,chemiluminescent reactions can occur in the presence of catalysts suchas alkaline phosphatase, horseradish peroxidase, metal ions or metalcomplexes. As such, a control siRNA can be labeled with achemiluminescent organic compound for transfection, wherein thetransfection efficacy is measured by chemiluminescence after thecatalyst is added to the contents of the transfected cell.

The use of labeled nucleotides is well known to persons of ordinaryskill, and labels such as enzymatic, mass, or radioactive labels, may beused in applications in which such types of labels would beadvantageous. Further descriptions of labels that are applicable fortransfection control siRNA in RTF testing protocols are found in U.S.Provisional Patent Application No. 60/542,646, 60/543,640, and60/572,270 and International Application PCT/US04/10343, wherein each isincorporated herein by reference.

The label can be attached directly to the control siRNA or through alinker. The label can be attached to any sense or antisense nucleotidewithin the siRNA, but it can be preferable for the coupling to bethrough the 3′ terminal nucleotide and/or 5′ terminal nucleotide. Aninternal label may be attached directly or indirectly through a linkerto a nucleotide at a 2′ position of the ribose group, or to anothersuitable position. For example, the label can be coupled to a5-aminoallyl uridine.

For example, linkers can comprise modified or unmodified nucleotides,nucleosides, polymers, sugars, carbohydrates, polyalkylenes such aspolyethylene glycols and polypropylene glycols, polyalcohols,polypropylenes, mixtures of ethylene and propylene glycols,polyalkylamines, polyamines such as polylysine and spermidine,polyesters such as poly(ethyl acrylate), polyphosphodiesters,aliphatics, and alkylenes. An example of a conjugate and its linker ischolesterol-TEG-phosphoramidite, wherein the cholesterol is theconjugate and the tetraethylene glycol (“TEG”) and phosphate serve aslinkers.

In one embodiment, the transfection control siRNA can be a type whereits presence is identified or measured by the effects on a cell. Thecontrol siRNA can be detected by an established and reproducible effectthat is caused by the presence of the control siRNA in a cell. Forexample, the control siRNA can be a type that causes cell toxicityand/or death when transfected into a cell, or can cause an identifiableor measurable response not related to RNAi. Cytotoxic siRNA can be usedas transfection control siRNA because the amount of cell toxicity can bedirectly related to the siRNA. The gene silencing efficacy of a testwell can be identified by the amount of toxicity induced by thecytotoxic siRNA in the control well. Also, the effect of the cytotoxicsiRNA can be compared to cells in blank wells as well as to cells intest wells. In the instance the cytotoxic siRNA induces cell death andthe cells in the blank wells or test wells do not show any toxiceffects, the transfection and/or gene silencing efficacy of the testwell can be compared to level of cell death induced by the cytotoxicsiRNA. However, established and reproducible toxic effects that arisefrom cytotoxic siRNA can be analyzed without being compared to cells inother wells.

In one embodiment, the transfection control siRNA is a cytotoxic siRNA.Some cytotoxic siRNA which have been identified include sensepolynucleotide sequences that include the following motifs: AAA; UUU;GCCA; or UGGC. Also, an siRNA can be toxic by including a longpolynucleotide sequence. The toxic control siRNA can include a longdsRNA that is about 50 base pairs, and preferably longer than 50 basepairs. Optionally, the toxic control siRNA can induce an interferonresponse that is toxic to the cells. Cytotoxic transfection controlsiRNA can be represented by Dharmacon's siCONTROL™ TOX. Additionally,siRNA that induce a toxic response through the RNAi pathway byinhibiting production of a protein vital to cell viability can be usedas cytotoxic siRNA.

B. Positive Control siRNA

Positive control siRNA can be well characterized siRNA that silence awell known gene. That is, the control siRNA silence a known gene withestablished and reproducible results. As such, the positive controlsiRNA can provide meaningful data regarding the efficacy of transfectionand/or gene silencing in a test plate having a similar RTF protocol orconditions. In part, this is because the control siRNA are known tosystematically silence a known gene to stop production of a knownprotein, wherein the known gene and known protein can be referred to asa control gene and control protein or control polypeptide, respectively.

Positive control siRNA can be distinguished from test siRNA by a numberof characteristics. Usually, test siRNA are being tested to identifywhether or not a target test gene will be silenced in an RTF protocol orcondition. On the other hand, positive control siRNA have well knownsequences and/or characteristics that systematically silence a wellknown gene in a reproducible and measurable manner. This allows thepositive control siRNA to provide meaningful results regarding theefficacy of the test siRNA because when the positive control gene is notsilenced or is overly silenced in the control well, the results obtainedfrom a test well from using the test siRNA may be unreliable. Examplesof positive control siRNA include siRNA that can silence MAP kinasegenes, glyceraldehyde-3-phosphate dehydrogenase (“GAPDH”) gene,cyclophilin B (“cyclo”) gene, Lamin A/C genes, and other wellestablished genes that can be silenced with siRNA having specificpolynucleotide sequences. Specific examples of positive control siRNAinclude Dharmacon's siCONTROL™ GAPD, siCONTROL™ Cyclophilin B, andsiCONTROL™ Lamin A/C.

Positive control siRNA can also include siRNA that inhibit the RNAipathway, which uses siRNA targeting RISC genes, Ago2 genes, eIF2C2genes, and the like for silencing. Additionally, these positive controlsiRNA can silence human, mouse, rat, or other animal control genes. Inaddition, positive control siRNA can target any gene that when silenced,gives a predictable result in e.g. a phenotypic

In one embodiment, the positive control siRNA can be selected tooptimize functionality in silencing the control gene. Preferably, thepositive control siRNA has between 50% and 100% gene silencingfunctionality with respect to the control gene, more preferably between70% and 100%, even more preferably between 80% and 100%, and mostpreferably between 90% and 100% functionality in silencing the controlgene.

Additionally, the positive control siRNA antisense strand can havevarying levels of complementarity with the control sequence to which ittargets (e.g., control mRNA or gene). Preferably, the antisense strandcan have about 50-100% complementarity with the target sequence, morepreferably, about 70-100% complementarity, even more preferably about80-100% complementarity, still even more preferably about 90-100%complementarity, and most preferably about 100% complementarity with thecontrol sequence.

In one aspect, the positive control siRNA can be selected by rationaldesign. As such, the positive control siRNA is selected using one ormore formulas that identify sequences that have desirable attributes andare more highly functional. Highly functional positive control siRNA canbe identified by rational design in order to perform more consistentlyand reproducibly than less functional siRNA under a wide range ofconditions found in RTF formats (e.g., cell densities).

In one embodiment, it can be preferable to select positive control siRNAthat have been previously identified from lists of siRNA that have beenselected using rational design algorithms. As such, the control siRNAcan be selected from Table I of the incorporated provisional applicationhaving Ser. No. 60/678,165. Table I is entitled “siGENOME Sequences forHuman siRNA,” and consists of columns “Gene Name,” “Accession No.,”“Sequence,” and “SEQ. ID NO.” Table I lists 92,448 19-mer siRNA sensestrand sequences, where antisense strand sequences were omitted forclarity. The siRNA sequences listed in Table I includes SEQ. ID NOs.1-92,448, wherein each preferably can also include a 3′ UU overhang onthe sense strand and/or on the antisense strand. Each of the 92,448sequences of Table I, when used in an siRNA, can also comprise a 5′phosphate on the antisense strand. Of the 92,448 sequences listed inTable I, 19,559 have an on-targeting set of modifications. A list ofsequences, identified by SEQ. ID NO., that have on-target modificationsis presented in Table II, entitled “List of Table I Sequences HavingOn-Target Modifications Identified by SEQ. ID NO.” On-targetmodifications are on SEQ. ID NOs. 1-22,300.

C. Negative Control siRNA

Negative control siRNA can be characterized as not being functionalwithin the RNAi pathway, and thereby do not induce gene silencing. Thiscan be accomplished by various mechanisms which include siRNA that arenot functional, siRNA that inhibit uptake and processing by RISC, and/orsiRNA that do not have complementarity to a gene or mRNA. For example,the negative control siRNA can fail to enter RISC. As such, negativecontrol siRNA can provide meaningful results that are well known andreproducible by not affecting the cellular processes of transfectedcells. Also, negative control siRNA can provide indications of genesilencing conditions and protocols by testing for any induced toxicity,loss of cellular function, or non-specific inhibition of proteinproduction.

In one embodiment, the negative control siRNA is non-functional siRNA,which does not function in the RNAi pathway. The non-functional siRNAcan provide an indication of transfection efficacy by monitoring theresponse of the transfected cell. Instances in which non-functionalsiRNA cause changes in gene expression, cell function, or phenotype maybe the result of factors other than RNAi mediated gene silencing, andcan provide an indication regarding the efficacy of gene silencing oftest wells having substantially similar RTF protocols and/or conditions.Non-functional siRNA can have certain sequences and/or chemicalmodifications in order to induce non-functionality. Additionally,non-functional siRNA can have a 17 base pair duplex or any duplex havingless than 18 base pairs. For example, non-functional siRNA can include a17 base pair duplex with 2′ modifications at the first and second sensenucleotides and on the first and second antisense nucleotides. Inanother example, a non-functional siRNA can include a 19 base pairduplex with 5′ deoxynucleotides on the 5′ end of the sense and theantisense strands.

In one embodiment, the negative control siRNA includes sequence and/ormodifications that inhibit uptake and processing by RISC. A negativecontrol siRNA can include a modification or size that inhibits beingtaken in and processed by RISC. That is, RISC is not able to perform afunction with mRNA having the sequence of the negative control siRNA,wherein the negative control has a modification or size that preventsbeing taken into RISC. This can be used to modify siRNA having selectedsequences that can be used for comparative purposes with test siRNA withthe same sequence but not having the modifications. As such, any genesilencing that arises from the negative control siRNA may result fromnon-specific silencing and provide an indication that the data obtainedfrom the test siRNA is not reliable. An example of the negative controlsiRNA that are not taken in or processed by RISC can include siCONTRO™RISC-Free (Dharmacon, Inc.).

For example, the negative control siRNA that are non-functional or arenot taken in and processed by RISC can have various 2′ modifications.This can include control siRNA that contain 2′ modifications on thefirst and second sense nucleotides, 2′ modifications on at least onethrough all pyrimidine sense nucleotides, 2′ modifications on the firstand/or second antisense nucleotides, 2′ modifications on at least onethrough all pyrimidine antisense nucleotides, and/or a 5′ carbon havinga phosphate modification at the sense or antisense 5′ terminalnucleotide. The 2′ modifications can be 2′-O-aliphatic modifications or2′-halogen modifications. The control siRNA can also includeinternucleotide modifications with phosphorothioates ormethylphosphonates.

The 2′ modifications can be characterized by as 2′-O-aliphaticmodifications such as 2′-O-alkyl modifications. For example, the2′-O-alkyl can be selected from the group consisting of 2′-O-methyl,2′-O-ethyl, 2′-O-propyl, 2′-O-isopropyl, 2′-O-butyl, 2′-O-isobutyl,2′-O-ethyl-O-methyl (i.e., —CH₂CH₂OCH₃), 2′-O-ethyl-OH (i.e.,—OCH₂CH₂OH), 2′-orthoester, 2′-ACE group orthoester, and combinationsthereof. Most preferably, the 2′-O-alkyl modification is a 2′-O-methylmoiety. Additionally, the 2′-halogen modifications can be selected fromthe group consisting fluorine, chlorine, bromine, or iodine; however,fluorine is preferred.

In one embodiment, the negative control siRNA can be an siRNA that has asense and/or antisense strand with limited or non-functionalcomplementarity to a gene or mRNA sequence. This can include siRNA thatare bioinformatically designed to minimize any potential targeting ofany known human or animal gene. The non-targeting negative control siRNAcan modified or unmodified, and examples include Dharmacon's siCONTRO™Non-Targeting siRNA #1 and siCONTROL™ Non-Targeting pool.

D. Dual Function Control siRNA

In one embodiment, the present invention includes dual function controlsiRNA. A dual function control siRNA can be used for two differentcontrol studies. This can include a single control siRNA that functionsboth as a transfection control and a positive control. Also, this caninclude a single control siRNA that functions both as a transfectioncontrol and a negative control. Examples of dual function controlsinclude any positive or negative control that includes a label, such asa fluorescent label (e.g., siGLO™ Cyclophilin B, siGLO™ Lamin A/C,siGLO™ RISC-Free, each from Dharmacon, Inc.).

E. Control siRNA Configurations

The control siRNA may be used individually (e.g., one siRNA sequence perwell) or as part of a pool. A pool of control siRNAs, as defined herein,refers to the use of at least two different control siRNAs within aspecific well, and usually at least four different control siRNAs. Eachcontrol siRNA can include between 18 and 31 base pairs, more preferablybetween 19 and 26 base pairs, and most preferably 19 and 21 base pairs.However, toxic control siRNA can have duplex regions with about 50 basepairs or greater than 50 base pairs, and some non-functional controlsiRNA can include less than 18 base pairs. Each control siRNA caninclude a sense strand and an antisense strand, which are preferably atleast substantially complementary to each other over the range of theduplex region. It is most preferable for the duplex region to have about100% complementarity.

Additionally, the control siRNA can have overhangs, bulges, mismatches,stability modifications, specificity modifications, hairpin structures,or other common features on target siRNA. Accordingly, additionalinformation regarding these features can be reviewed in thecross-referenced patent application filed herewith having AttorneyDocket No. 16542.1.1, entitled APPARATUS AND SYSTEM HAVING DRY GENESILENCING COMPOSITIONS, with Barbara Robertson, Ph.D., et al. asinventors.

A reduction in off-targeting or increased specificity can also beachieved by using control siRNA concentrations that are below the levelthat induces off-target effects. As an example, transfection of a singlecontrol siRNA at 100 nM can induce 90% silencing, yet the highconcentration of the siRNA may also induce off-target effects. Incontrast, a pool of four control siRNAs (e.g., total concentration of100 nM, 25 nM each) can similarly induce 90% silencing. Since each siRNAis at a four-fold lower concentration, the total number of off-targetsis fewer. Thus, in order to obtain control gene silencing with inhibitedor no off-target effects, a highly functional siRNA can be used at lowconcentrations, or pools of control siRNA targeting the same gene can beused with each siRNA of the control pool having a concentration that issufficiently low to minimize off-target effects. Preferably, the totalamount of control siRNAs can be delivered at concentrations that areless than or equal to about 100 nM, more preferably less than or equalto about 50 nM, even more preferably less than or equal to about 25 nM,and most preferably less than or equal to about 10 nM.

VII. Polynucleotide Carriers

In one embodiment, the present invention includes polynucleotidecarriers that can interact with a control siRNA, and transport thecontrol siRNA across a cell membrane. However, in other embodiments ofthe invention modes of transfection can be implemented without carriers,such as by electrophoresis, precipitation, particle bombardment,optoporation, and microinjection. Usually, polynucleotide carriersinclude a positive charge that interacts with the negatively chargedphosphates on the polynucleotide backbone. Polynucleotide carriers arewell known in the art of cellular nucleic acid deliver. Preferredpolynucleotide carriers include polymers, lipids, lipopolymers,lipid-peptide mixtures, and the like that are capable of complexing withan siRNA and delivering the siRNA into a cell in a manner that retainsthe gene silencing functionality without being overly toxic. As such,routine experimentation can be implemented with procedures describedherein with respect to optimizing RTF protocols in order to identify theoptimal polynucleotide carrier for a certain system or cell.

In one embodiment, lipids or lipid-peptide mixtures are preferable forintroducing siRNA into a target cell. Typically, the lipid is a cationiclipid. Cationic lipids that can be used to introduce siRNA into cellscan be characterized by having little or no toxicity (e.g., defined asless than 15-20% toxicity), which can be measured by AlamarBlue orequivalent cell viability assays. However, not all lipids arefunctionally equivalent and certain lipids can perform better withspecific cell lines. Thus, the foregoing optimization procedures can beemployed to determine an appropriate lipid and lipid concentration fordelivering siRNA for a specific cell line. Peptides that have affinityto one or more proteins, lipids, lipid-polysaccharide, or othercomponents of the cell membrane can be conjugated to the siRNA and usedindependent of lipids or advantageously combined with one or more lipidsto form a polynucleotide carrier. Such lipid-peptide mixtures canenhance RTF of siRNA. Cholesterol conjugates can be similarly coupled tothe siRNA and be used independent of polynucleotide carriers oradvantageously combined therewith.

Briefly, in order to identify whether a given lipid is acceptable forsiRNA RTF testing protocols, two or more well characterized controlsiRNAs can be tested under a variety of lipid, media, and siRNAconcentrations using the optimizing protocols described herein.Subsequently, the level of transfection and/or gene silencing and/or thelevel of cell death are quantified using art-accepted techniques.Suitable lipids for siRNA RTF testing protocols include OLIGOFECTAMINE™,TransIT-TKO™, or TBIO Lipid 6™, LIPOFECTAMINE™ 2000, lipids DharmaFECT™1, DharmaFECT™ 2, DharmaFECT™ 3, and DharmaFECT™ 4 (Dharmacon, Inc.).The term “DharmaFECT™” (followed by any of the numerals 1, 2, 3, or 4)or the phrase “DharmaFECT™ transfection reagent,” refers to one or morelipid-based transfection reagents that have been optimized to transfectsiRNA rather than larger nucleic acids (e.g., plasmids). Additionalinformation on lipids can be obtained in U.S. Pat. Nos. 5,674,108,5,834,439, 6,110,916, 6,399,663, and 6,716,582, and internationalpublications WO 00/12454 and WO 97/42819, wherein each is incorporatedherein by reference.

The formation of a functional control siRNA-lipid complex can beprepared by combining control siRNA and the lipid. As such, anappropriate volume of lipid at a selected concentration can be combinedwith a volume of media and/or buffer to form a lipid-media orlipid-buffer having a suitable concentration of lipid. For example, avolume of lipid media ranging from about 5-50 microliter (“uL”) caninclude about 0.03-2 micrograms (“ug”) of lipid to be introduced intoeach well of a 96-well plate, and the amount of lipid can be changed tocorrespond with other well sizes. The choice of media and/or buffer canimprove the efficiency of the RTF protocol. Some media contain one ormore additives that induce cell toxicity and/or non-specific genemodulation during RTF testing protocols. Examples of preferred media orbuffers include Opti-MEM™ (GIBCO, Cat. #31985-070), HyQ-MEM-RS™(HyClone, Cat.#SH30564.01), Hanks Balanced Salt Solution™, or equivalentmedia. A suitable media can be identified by employing the optimizationprotocol described herein.

The lipid-media or lipid-buffer can be introduced into a well by avariety of methods including hand-held single and multi-channelpipettes, or more advanced and automated delivery systems that caninject measured volumes of the lipid solution into a well. The lipidsolution can be incubated in the well that contains the dried controlcomposition for a period of time that is sufficient to solubilize orsuspend the siRNA, and to form control complexes. In general, theprocess of siRNA control solubilization and complex formation canrequire about 20 minutes, but usually not more than 120 minutes. Thecomplex formation process is generally performed at room temperature,but can be performed at temperatures ranging from 4-37° C. In someinstances, the lipid and control siRNA can be mixed by agitating theplate (e.g., swirl, vortex, sonicate) for brief periods (e.g.;seconds—minutes) to enhance the rate of control siRNA solubilization andcomplex formation.

Additionally, any of the foregoing polynucleotide carriers can beincluded in systems or kits in accordance with the present invention.Such kits can include the plates having control compositions, and can bedistributed with siRNA solubilizing or suspending solutions,polynucleotide carriers, carrier solutions, reagents, cell media, andthe like.

VIII. Well Arrangements

In one embodiment, the siRNA RTF testing plates that include multiplewells having different dry control compositions can have the wellsorganized into predefined arrangements. Such arrangements can correspondto the type of assay being employed with the siRNA RTF testing plate.That is, when a family of genes is being studied, a first control siRNAcan be organized in one column or row while a second control ortargeting siRNA can be organized in a different column or row. Thus, thewells can be organized into a pre-selected arrangement so thatparticular control siRNAs are in a pre-selected pattern on a plate. Thepre-selected pattern can include various control wells, such as thosethat include one or more negative, positive, and/or transfectioncontrols. Also, the pre-selected pattern can include wells that areempty or substantially devoid of any siRNA, which can be used ascontrols and for calibrations.

It can be beneficial to have control siRNAs that are pre-dried incorresponding wells of different well plates so that multiple RTFtesting plates can be prepared simultaneously. This can allow for RTFtesting plates to have gene silencing compositions at standardizedpositions and amounts of control siRNAs, which is beneficial for usingstandardized well plates in multiple experiments that can be conductedover time without introducing variability between the plates. The use ofstandardized plate arrangements can provide a series of plates that canbe used over time and provide data that can be analyzed together.

For example, a plate comprising a plurality of columns of wells caninclude a transfection control in the first column, positive controlsfor RNAi in the second column, negative controls for RNAi in a thirdcolumn, a pool of siRNAs directed against a single target in a fourthcolumn, and individual members of the siRNA pool that comprise thefourth column are in subsequent columns, such as the fifth throughtwelfth columns. Alternatively, the fifth through twelfth columns cancomprise different concentrations of each siRNA in the pool of thefourth column, with the amount of siRNA increasing from well to well ordecreasing from well to well. Each well can include one concentration ofeach siRNA in the pool, or two, three, four, five, or moreconcentrations of each siRNA in the pool can be in different wells. Thenumber of concentrations of siRNA that can be used is limited only bythe number of wells on the plate; however, multiple plates can beconfigured to be used together with a predefined pattern that spreadsacross all the plates. Additionally, the pre-selected patterns ofcontrol siRNA concentration gradients can be used as a pattern that canbe observed so that the optimal amount of each control siRNA can bedetermined by observing the level of transfection and/or gene silencingnumber of concentrations of that particular control siRNA.

FIGS. 1A and 1B illustrate embodiments of plate arrangements similarwith the foregoing concentrations arrangements. While the wells areshown to be square, it should be recognized that they can be any shape.Also, the well plate can include any number of wells, and the number ofwells depicted is merely for example. In the figures the wells aredefined as follows: “Tc” indicates a transfection control well, whereinthe increasing corresponding numbers identify different transfectioncontrols; blank wells indicate wells devoid or substantially devoid ofany siRNA; “+” indicates a positive control; “−” indicates negativecontrols; “P1” through “P1_(N)” indicate a first pool which silences afirst gene at a concentration gradient; “P2” through “P2_(N)” indicate asecond pool which silences a second gene at a concentration gradient;“1A” through “1_(N)” indicate a first individual test siRNA of the firstpool at a concentration gradient; “2A” through “2_(N)” indicate a secondindividual test siRNA of the first pool at a concentration gradient;“3A” through “3_(N)” indicate a third individual test siRNA of the firstpool at a concentration gradient; “4A” through “4_(N)” indicate a firstindividual test siRNA of the second pool at a concentration gradient;“5A” through “5_(N)” indicate a a second individual test siRNA of thesecond pool at a concentration gradient; and “6A” through “6_(N)”indicate a third individual test siRNA of the second pool at aconcentration gradient. Thus, FIG. 1A illustrates a well plate assayinga single pool, and FIG. 1B illustrates a well plate assaying multiplepools. Additionally, a well plate can include more than two pools.

FIG. 2A. is a schematic diagram that illustrates an embodiment of a wellplate having control siRNA. More particularly, wells A1-H1, G2, and H2are blank wells devoid of siRNA. For example, wells A1-H1 are availablefor RTF testing controls. Well G2 can be used as a mock transfectioncontrol, and well H2 can be an untreated cell control. Wells A2-F2contain negative and positive transfection control siRNAs. Moreparticularly, the control wells include the following: well A2 includesa non-targeting negative control siRNA (e.g., siCONTROL™ Non-TargetingsiRNA pool from Dharmacon, Inc.); well B2 includes an siRNA thatinhibits being taken in and processed by RISC (e.g., siCONTRO™ RISC-Freefrom Dharmacon, Inc.); well C2 includes a dual control siRNA that can beused as a negative control and a transfection control (e.g., siGLO™RISC-Free from Dharmacon, Inc.); well D2 includes a positive controlsiRNA targeting GAPDH control gene (e.g, siCONTROL™ GAPD from Dharmacon,Inc.); well E2 includes a positive control siRNA targeting cyclophilin Bcontrol gene (e.g., siCONTRO™ Cyclophilin B siRNA from Dharmacon, Inc.);and well F2 is a positive control siRNA targeting Lamin A/C control gene(e.g., siCONTRO™ Lamin A/C from Dharmacon). The wells in columns 3-12can each contain a test siRNA or test siRNA pool such as any ofDharmacon's SMARTpool™ siRNA reagents. Alternatively, well F2 can be auser defined control siRNA.

FIG. 2B is a schematic diagram that illustrates an embodiment of anoptimization well plate or an RTF testing plate having control siRNA. Asshown, rows A-C can contain three negative control siRNAs (e.g.,siCONTRO™ Non-Targeting siRNA pool, siCONTROL™ RISC-Free siRNA, siGLO™RISC-Free siRNA, each from Dharmacon, Inc.). Rows D-E can contain threepositive transfection control siRNAs (e.g., siCONTROL™ GAPD, siCONTROL™Cyclophilin B siRNA, siCONTROL™ Lamin A/C siRNA, each from Dharmacon,Inc.). Alternatively, Row F can be a user defined control siRNA. Row Gdoes not contain siRNA and can be used for mock-transfected cells inorder to study the effect of the transfection reagent alone on cellviability and/or mRNA expression. Row H does not contain siRNA and iscan be used for untreated cells, and can serve as a 100% viabilitycontrol and/or 100% mRNA level control.

FIG. 2C is a schematic diagram that illustrates an embodiment of anoptimization plate or an RTF testing plate having control siRNA. Thetesting plate includes an arrangement of wells that can be used in aprocedure for optimizing RTF conditions. As shown, column 1 includesblank wells devoid of siRNA, and can be used as a blank that does notreceive cells. This column can be used for a standard curve or otherexperimental controls that may be desired for an RTF testing protocolfor assessing the efficacy of gene silencing. Column 2 includes blankwells devoid of siRNA, and can be used as a blank that does receivecells. This column can be used as an untreated reference for differentvolumes of polynucleotide carrier, such as DharmaFECT™ transfectionreagent, which is tested in columns 3-12. Columns 3-7 can containnegative control siRNAs, which serve as negative control samples foreach DharmaFECT™ transfection reagent volume used. As shown, up to fivevolumes of each DharmaFECT™ transfection reagent may be tested in oneplate. In each row, the DharmaFECT™ transfection reagent volumes incolumns 3-7 can be repeated in columns 8-12. This can allow the negativecontrol siRNAs to serve as references for any gene silencing that occursin the wells of columns 8-12, which contain positive transfectioncontrol siRNAs. Additionally, rows A-D can be seeded with a low celldensity, and rows E-H can be seeded with a high cell density. Thus, theplate can include a combination of different RTF conditions, which canbe used to determine the optimal amount of DharmaFECT™ transfectionreagent, DharmaFECT™ transfection reagent volume, and cell number.

FIG. 2D is a schematic diagram that illustrates an embodiment of anoptimization well plate or an RTF testing plate having control siRNA. Asshown, the plate is arranged with blanks, negative control siRNA, andpositive control siRNA as in FIG. 2C. However, this plate can be assayedwith different polynucleotide carrier concentrations. Rows A-D can beused with low cell densities with DharmaFECT™ transfection reagent atvolumes ranging from 0.03 uL/well (e.g., columns 3 and 8) to 0.5 uL/well(e.g., columns 7 and 12). Rows E-H can be used with high cell densitieswith DharmaFECT™ transfection reagent at volumes ranging from 0.06uL/well (e.g., columns 3 and 8) to 1.0 uL/well (e.g., columns 7 and 12).

EXAMPLES

The following examples are provided to describe some embodiments of thepresent invention in a manner that can be use by one of skill in the artto practice the present invention. Additionally, the following examplesinclude experiments that were actually performed as well as propheticexperiments. Additional examples and supplementary information for thefollowing examples can be reviewed in the incorporated references havingAttorney Docket No. 16542.1.1, entitled APPARATUS AND SYSTEM HAVING DRYGENE SILENCING COMPOSITIONS, with Barbara Robertson, Ph.D., et al. asinventors, Attorney Docket No. 16542.1.2, entitled APPARATUS AND SYSTEMHAVING DRY GENE SILENCING POOLS, with Barbara Robertson, Ph.D., et al.as inventors, and U.S. Provisional Application Ser. No. 60/678,165. Thepolynucleotide sequences that were used in the examples can be found inTables I-IV of U.S. Provisional Application Ser. No. 60/678,165, and thesequence listing of the reference having Attorney Docket No. 16542.1.1,entitled APPARATUS AND SYSTEM HAVING DRY GENE SILENCING COMPOSITIONS,with Barbara Robertson, Ph.D., et al. as inventors.

Example 1

The effect of plate conditions on RTF protocols was assayed in order todetermine optimum conditions. The different types of plate coatings thatwere studied included untreated, fibronectin-treated, poly-L-Lysinetreated, MATRIGEL™-treated, and CELLBIND™ plates. In the poly-L-lysineplates, each well was treated with 50 uL of 5 ug/uL poly-L-lysine for 1hour, and washed with ddH₂O (3×) and dried under a UV light for 20minutes. MATRIGEL™ plates were obtained from BD Biosciences (Catalog No.354607, Bedford, Mass.). Fibronectin plates were purchased from BectonDickinson Labware (Biocoat cellware, Catalog No. 354409, Bedford, Mass.)and CELLBIND™ plates were obtained from Corning (Catalog No. 3300). Foreach study, varying amounts of human cyclophilin B siRNA (e.g., cyclo3)were added to different wells at 0-250 nM.

The results of these studies are provided in FIGS. 3A-3J, which aregraphs illustrating the efficacy of the RTF testing procedures. Thegraph of FIG. 3A depicts the cell viability of the untreated plates,wherein the cell viability drops fairly steadily as the lipidconcentration increases. At low lipid concentrations (e.g. 0.125 uglipid per 100 uL) the cells were sufficiently viable to providemeaningful gene silencing results. The graph of FIG. 3B depicts thecyclophilin B siRNA successfully silenced the target gene. The graph ofFIG. 3C depicts the cell viability of polylysine plates to similarlydecrease as the lipid concentration increase, and the lower lipidconcentrations were not overly toxic to the cells. The graph of FIG. 3Ddepicts the cyclophilin B siRNA successfully silenced the target gene.The graph in FIG. 3E depicts the CELLBIND™ plates to have reducedtoxicity with acceptable levels of cell viability being preserved up to0.25 ug of lipid per 100 uL. However, the graph of FIG. 3F shows thatgene silencing was only moderate, and was determined to be unacceptable.The graph in FIG. 3G depicts the MATRIGEL™ plates to have overall poorcell viability under all conditions, and the graph of FIG. 3H wasconsidered to be unreliable. The graph of FIG. 31 depicts thefibronectin-coated plates to also have poor cell viability, and thegraph of FIG. 3J was considered to be unreliable.

Example 2

The optimization of RTF protocols to induce gene silencing was studiedby assessing the siRNA functionality in relation to cell density. ThesiRNAs of varying functionalities (e.g., F50-F95) were reversetransfected under a range of cell densities (e.g., 10,000-40,000 cellsper well) using lipid concentrations that induced minimal levels of celltoxicity (0.063 ug of lipid per 100 uL for 10,000 cells per well, 0.125ug of lipid per 100 uL for 20,000 cells per well, and 0.25 ug of lipidper 100 uL for 40,000 cells per well). The study was performed withcontrol cyclophilin B siRNA (e.g., cyclo3, cyclo28, and cyclo37 withfunctionalities of 95, 75, and 50, respectively).

FIG. 4 is a graphical representation of the gene silencing of increasingconcentrations of siRNA in relation to the increasing number of cells.The graph shows that highly functional siRNA induced greater genesilencing under a broad range of conditions. The cyclo3 siRNA induced60% or more silencing at all three cell concentrations. In contrast,less functional molecules (e.g., cyclo 28) performed well at only lowcell densities (e.g., 10,000 cells per well), but less well at highercell densities (e.g., 20-40,000 cells per well). Thus, increasedfunctional siRNA, which are rationally designed greatly improve genesilencing.

Example 3

A population of randomly selected siRNAs derived from a walk targetingDBI (e.g., NM_(—)020548, position 202-291) was assessed for the abilityto induce toxicity. The collection of siRNAs consisted of 90 individual(e.g., 19 bp) duplexes, and covered the respective regions in singlebase steps. Duplexes were forward transfected into HeLa cells usingLIPOFECTAMINE™ 2000, and a threshold of 75% cell viability was used asthe cutoff to distinguish toxic from nontoxic sequences.

FIG. 5A is a graphical representation of the results of the toxicitystudy. As shown, the siRNAs transfected under these conditions wereobserved to induce varying levels of cellular toxicity. Overall, 14 outof 90 siRNA duplexes (e.g., 15.5%) were found to decrease cellularviability below 75%, which is identified by the horizontal dashed line.These toxic siRNA can be identified by the numbers within the boxes thatshow cell survival below the dashed line, and can be used astransfection control siRNA

FIG. 5B is a graphical representation of an the cell toxicity andviability obtained from individual siRNAs of 48 functional (e.g., >70%silencing) pools of four siRNA targeting 12 different genes. Only twelveof the forty-eight sequences (e.g., 25%) decreased cellular viabilitybelow 75%.

FIG. 5C is a graphical representation of the toxicity of exemplary siRNAof the pools depicted in FIG. 5B. While all eight duplexes targetingMAP2K1 and MAP2K2 show greater than 80% gene silencing, only a singlesiRNA in each quartet reduces cell viability below 75% (e.g., MAP2K1-d4and MAP2K2-d3). Thus, as the remaining siRNAs in each group were equallyfunctional, but non-toxic, the toxicity induced by MAP2K1-d4 andMAP2K2-d3 is unrelated to target knockdown.

A linear display of the distribution of toxic siRNA along the DBI walkshowed that the dispersal of these toxic sequences was frequentlynon-random (i.e., clustered) and suggested the presence of one or moremotifs that were responsible for the observed toxicity (e.g., see boxedareas of FIG. 5A). Subsequent analysis of the toxic sequences from therandom functional siRNA set revealed that all twelve sequences containedeither an AAA/UUU or GCCA/UGGC motif. To determine whether a correlationexisted between the motifs and toxicity, three additional, randomlyselected, groups of siRNA that contained either AAA/UUU motifs,GCCA/UGGC motifs, or neither motif, were chosen and tested for theability to induce cell death. FIGS. 6A-6C are graphical representationsthat shown the siRNA containing the AAA/UUU and GCCA/UGGC motifsexhibited a higher probability of inducing toxicity (e.g., 56% and 53%,respectively) in comparison with the non-motif siRNA (e.g., 6%). Sincethe statistical analysis (e.g., T-Test) p-value was 1.3×10⁻⁷ for thesetwo samples, the results show a strong correlation exists between siRNAinduced cellular toxicity and delivery of duplexes containing theAAA/UUU or GCCA/UGGC motifs. Thus, siRNA having toxic motifs can be usedas transfection controls.

Example 4

The ability of siRNA having a toxic motif to induce cell death wasstudied while the RNAi pathway was severely compromised. Previousstudies revealed that eIF2C2/hAgo2 is required for mRNA cleavage, andthat silencing of these gene products can severely cripple the RNAipathway. In a control study shown in FIG. 11A, a first set of HeLa cellswere forward transfected with siRNA directed against eIF2C2 (e.g.,siRNA-eIF2C2), and a second set were transfected with control siRNA(e.g., siRNA-RISC-Free) that are not processed by RISC in T1. Each setof the HeLa cells where then transfected with siRNA-RF (i.e.,siRNA-RISC-Free) and pEGFP in T2.

The results of the control study of FIG. 7A can be viewed in the imagesdepicted in FIGS. 7B-7I. The results shown in FIGS. 7B and 7C show thatthe siRNA-RISC free does not inhibit EGFP expression from pEGFP.However, FIGS. 7D and 7E show that the siRNA-EGFP was able to inhibitproduction of the green fluorescent protein when the RNAi pathway wasnot compromised. The results in 7F and 7G show that the even ifsiRNA-eIF2C2 inhibited the functionality of the RNAi pathway, thecontrol siRNA-RF did not have any effect because the cells expressed thegreen fluorescent protein. Additionally, FIGS. 7H and 7I show that thesiRNA-eIF2C2 inhibited the functionality of the RNAi pathway because thepEGFP show the cells were transfected and expressed the greenfluorescent protein in the presence of siRNA-EGFP.

The importance of the RNAi pathway in siRNA-induced cell death wastested by transfecting HeLa with the siRNA-eIF2C2 and control siRNA-RF(T1 of FIG. 7A Experiment). The HeLa cells were subsequently transfectedwith toxic siRNA containing either the AAA/UUU or GCCA/UGG motifs (T2 ofFIG. 7A Experiment). FIG. 7J is a graphical representation of the genesilencing obtained with siRNA-eIF2C2, which eliminated the ability oftoxic siRNA to induce cell death. As parallel experiments, cells werepre-transfected with control siRNA-RF (T1) that exhibited toxicitycharacteristic of these sequences, and it was concluded that an intactRNAi pathway was necessary for siRNA-induced toxicity.

Example 5

Additional experiments were conducted to determine the involvement ofthe RNAi pathway in siRNA induced toxicity. The ability of toxic siRNAto induce cell death was tested with siRNA having 19 and 17 base pairs.Previous studies have shown that duplexes that are shorter than 19 basepairs targeted mRNA sequences inefficiently, which suggests that Dicerand/or RISC fail to mediate RNAi when duplex sequence length drops tosome level below 19 base pairs.

FIG. 8 is a graphical representation of the results of toxic siRNAhaving 19 base pairs and corresponding siRNA having 17 base pairs. Asthe graph illustrates, the siRNA having 17 base pairs resulted insignificantly less toxicity. This suggests that entry and/or processingby RISC is necessary for siRNA to induce toxicity. Thus, preferably thetoxic control siRNA is at least 18 base pairs, and more preferably atleast 19 base pairs. Together these results demonstrate that siRNAinduced off-target effects can generate measurable phenotypes.

Example 6

Three different lipids were studied on two different cell lines tooptimize an RTF protocol. Accordingly, positive control siRNAs (e.g.,cyclo3) at various concentrations were dried on well floors of 96-wellplates such that final concentrations varied between 0 and 250 nM. Lipidsolutions of OLIGOFECTAMINE™, DharmaFECT™ 1, or TBio were mixed withOpti-MEM™ and tested on 10,000 A549 cells, or 5,000 3T3L1 cells.

FIG. 9A is a graphical representation of the toxicity of the lipid inthe A549 cells. OLIGOFECTAMINE™ and TBio induced minimal toxicity at allconcentrations. DharmaFECT™ 1 produced minimal toxicity at 0.125 ug oflipid per 100 uL. FIG. 9B is a graphical representation of the genesilencing efficacy achieved with the lipids. OLIGOFECTAMINE™ was shownto have inefficient gene silencing so as to be not suitable for use insiRNA RTF of A549 cells. DharmaFECT™ 1 and TBio provided excellent genesilencing at all amounts of siRNA, and were suitable for use in A549cells.

FIG. 10A is a graphical representation of the toxicity of the lipid inthe cells. OLIGOFECTAMINE™ and TBio induced minimal toxicity at allconcentrations. DharmaFECT™ 1 produced minimal toxicity at 0.125 ug oflipid per 100 uL. FIG. 10B is a graphical representation of the genesilencing efficacy achieved with the lipids. OLIGOFECTAMINE™ and Tbioeach showed inefficient gene silencing so as to be not suitable for usein siRNA RTF of 3T3L1 cells. DharmaFECT™ 1 provided excellent genesilencing at all amounts of siRNA, and were suitable for use in 3T3L1cells.

Example 7

The optimization of reagents that induce the least amount of celltoxicity and death were studied in a RTF test protocol with threeseparate lipid-media or lipid-buffer mixtures. Control siRNA (e.g.,cyclo3) at various concentrations were used with lipid solutions ofDharmaFECT™ 1-Opti-MEM™, DharmaFECT™ 1-HyQ-MEM™, or DharmaFECT™ 1-HanksBalanced Salt Solution (“HBSS”).

FIG. 11A is a graphical representation of the gene silencing obtainedwith the foregoing lipid solutions. The gene silencing in each culturewas shown to be very similar (e.g., >80% silencing. FIG. 11B is agraphical representation of the toxicity obtained with the foregoinglipid solutions. The toxicity varied depending on the lipid and mediaconditions. At 0.125 ug of lipid per 100 uL, both HyQ-MEM™ and HBSSperformed more consistently than Opti-MEM™ with nearly 100% cellviability for HyQ-MEM™ and HBSS compared to about 80% viability forOpti-MEM™. At higher lipid concentrations of 0.25 ug per 100 uL thedifferences in the performance of Opti-MEM™, HyQ-MEM™ and HBSS are moresignificant. At 0.25 ug of lipid per 100 uL, cell viability withDharmaFECT™ 1-Opti-MEM™ solutions was approximately 60%, 75%, 50%, 50%,and 25% for plates that had been aged 1, 2, 3, 4, and 8 weeks,respectively. In contrast, cell viability of cultures treated withDharmaFEC™ 1-HyQ-MEM™ and DharmaFECT™ 1-HBSS were greater than 90%.These results identify HyQ-MEM™ and HBSS as preferred reagents for siRNARTF protocols due to greater consistency across lipid concentrations.

Example 8

In one example, a RTF testing plate or series of plates can be designedin order to optimize RTF with control siRNA. Accordingly, the plates canbe configured to include any of the following variables: (1) theconcentration of individual or pools of control siRNA can be between0.01-250 nM, more preferably between 0.05 and 100 nM, even morepreferably between 0.1 and 50 nM, still even more preferably between 0.5and 25 nM, and most preferably between 0.75 and 10 nM or about 1 nM; (3)the types of polynucleotide carrier can be a lipid such as DharmaFECT™1, DharmaFECT™ 2, DharmaFECT™ 3, or DharmaFECT™ 4; (3) the concentrationof the lipid polynucleotide carrier can be at concentrations of 0.05-1ug per 100 uL of solution, more preferably at concentrations of 0.05-0.5ug of lipid per 100 uL of solution, even more preferably still atconcentrations of 0.05-0.25 ug of lipid per 100 uL of solution, and mostpreferably at concentrations of 0.05-0.1 ug per 100 uL of solution; (4)the types of media and/or buffer used to complex the lipid beingpreferably Opti-MEM™, more preferably HyQ-MEM™, and most preferablybuffered salt solutions such as Hanks Buffered salt solution orequivalent mixtures; and (5) the types and amounts of cells havingdensities of 1,000 to 35,000 cells per 0.35 cm² preferred densities of2,000-30,000 cells per 0.35 cm², more preferably 2,000-20,000 cells per0.35 cm², even more preferably 2,000-15,000 cells per 0.35 cm², and mostpreferably cell densities of 2,000-10,000 cells per 0.35 cm².

The present invention may be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments are to be considered in all respects only as illustrativeand not restrictive. The scope of the invention is, therefore, indicatedby the appended claims rather than by the foregoing description. Allchanges which come within the meaning and range of equivalency of theclaims are to be embraced within their scope.

1. A reverse transfection plate for testing the efficacy of genesilencing, the plate comprising: at least a first control well includinga substantially dry first control composition having at least a firstcontrol siRNA capable of providing a first indication of gene silencingefficacy, the first control composition being configured such that theat least first control siRNA is capable of being solubilized orsuspended in an aqueous medium in an amount sufficient for transfectingcells in the first control well.
 2. A plate as in claim 1, whereincontrol siRNA is at least one of a transfection control siRNA, positivecontrol siRNA, or negative control siRNA.
 3. A plate as in claim 2,further comprising at least a second control well including asubstantially dry second control composition having at least a secondcontrol siRNA capable of providing a second indication of gene silencingefficacy that is different from the first indication, the second controlcomposition being configured such that the at least second control siRNAis capable of being solubilized or suspended in an aqueous medium in anamount sufficient for transfecting cells in the second control well. 4.A plate as in claim 2, wherein the positive control siRNA silencesexpression of a known gene.
 5. A plate as in claim 4, wherein thepositive control siRNA silences expression of at least one of acyclophilin B, lamin A/C, or glyceraldehyde-3-phosphate dehydrogenase.6. A plate as in claim 2, wherein the transfection control siRNAincludes a label.
 7. A plate as in claim 6, wherein the label is coupledwith a 5′ terminal nucleotide or 3′ terminal nucleotide on one of asense strand or an antisense strand.
 8. A plate as in claim 7, whereinthe label is a fluorescent label on the sense strand.
 9. A plate as inclaim 2, wherein the transfection control siRNA is toxic to cells.
 10. Aplate as in claim 2, wherein the negative control siRNA isnon-functional siRNA.
 11. A plate as in claim 10, wherein thenon-functional siRNA includes at least one of a 17 base pair duplexcontaining a 17 base pair duplex having 2′ modifications on a first andsecond sense nucleotide, and having 2′ modifications at the first andsecond antisense nucleotide, or a 19 base pair duplex having 5′ deoxynucleotides on sense and antisense 5′ terminal nucleotides.
 12. A plateas in claim 2, wherein the negative control siRNA inhibits being takenin and processed by RISC.
 13. A plate as in claim 3, wherein the firstcontrol composition includes a positive control siRNA and the secondcontrol composition includes a negative control siRNA.
 14. A plate as inclaim 3, wherein the first control composition includes a pool ofcontrol siRNAs.
 15. A plate as in claim 3, further comprising at least athird control well including a substantially dry third controlcomposition having at least a third control siRNA capable of providing athird indication of gene silencing efficacy that is different from atleast one of the first indication or second indication, the thirdcontrol composition being configured such that the at least thirdcontrol siRNA is capable of being solubilized or suspended in an aqueousmedium in an amount sufficient for transfecting cells in the thirdcontrol well.
 16. A plate as in claim 1, further comprising at least onewell devoid of siRNA.
 17. A plate as in claim 1, wherein a total amountof control siRNA in the first control composition is present in anamount for transfecting cells in only the first control well.
 18. Aplate as in claim 1, further comprising at least one well including asubstantially dry gene silencing composition, the gene silencingcomposition having at least a first test siRNA which silences a firsttarget gene, the gene silencing composition being configured such thatthe at least first test siRNA is capable of being solubilized orsuspended in an aqueous medium in an amount sufficient for transfectingcells in the well, wherein the gene silencing composition ischaracterized by at least one of the following: the at least first testsiRNA is not modified; the at least first test siRNA has a stabilizingmodification; the at least first test siRNA has a modification to limitoff-targeting; the at least first test siRNA has a conjugate; or the atleast first test siRNA has a hairpin structure.
 19. A reversetransfection system for testing the efficacy of gene silencing, thesystem comprising: a plate comprising at least a first control wellincluding a substantially dry first control composition having at leasta first control siRNA, wherein the at least first control siRNA iscapable of being solubilized or suspended in an aqueous medium in anamount sufficient for transfecting cells in the well; and apolynucleotide carrier configured to complex with the at least firstcontrol siRNA.
 20. A system as in claim 19, wherein the control siRNA ischaracterized by one of the following: a positive control siRNA thatsilences expression of a known gene; a transfection control siRNA thatincludes a fluorescent label; a transfection control siRNA that includesat least one toxic motif; a negative control siRNA that isnon-functional siRNA; or a negative control siRNA that inhibits beingtaken in and processed by RISC.
 21. A system as in claim 20, furthercomprising at least a second control well including a substantially drysecond control composition having at least a second control siRNA,wherein the at least second control siRNA is capable of beingsolubilized or suspended in an aqueous medium in an amount sufficientfor transfecting cells in the well.
 22. A system as in claim 21, furthercomprising at least a third control well including a substantially drythird control composition having at least a third control siRNA, whereinthe at least third control siRNA is capable of being solubilized orsuspended in an aqueous medium in an amount sufficient for transfectingcells in the well.
 23. A system as in claim 21, further comprising atleast one well devoid of siRNA.
 24. A method of testing the efficacy ofgene silencing with control siRNA, the method comprising: adding anaqueous medium to a first control well in a well plate, wherein thefirst control well includes a first control siRNA; solubilizing orsuspending the first control siRNA in the aqueous medium; adding cellsto the first well under conditions that permit transfection; anddetermining the effect of the first control siRNA on the cells.
 25. Amethod as in claim 24, wherein the aqueous medium includes apolynucleotide carrier, and further comprising: forming a complex withthe first control siRNA and the polynucleotide carrier, wherein thecomplex is suspended or solubilized in the solution; and contacting thecomplex to a cell within the first control well.
 26. A method as inclaim 24, wherein the control siRNA is characterized by at least one ofthe following: a positive control siRNA that silences expression ofknown genes; a transfection control siRNA that includes a fluorescentlabel; a transfection second control siRNA is toxic to cells; a negativecontrol siRNA that is non-functional siRNA; or a negative control siRNAconfigured to inhibit being taken in and processed by RISC.
 27. A methodas in claim 26, further comprising: adding the aqueous medium to asecond control well in the well plate, wherein the second control wellincludes a second control siRNA; adding cells to the second control wellunder conditions that permit transfection; and determining the effect ofthe second control siRNA on the cells.
 28. A method as in claim 27,further comprising: adding the aqueous medium to a third control well inthe well plate, wherein the third control well includes a third controlsiRNA; adding cells to the third control well under conditions thatpermit transfection; and determining the effect of the third controlsiRNA on the cells.
 29. A method as in claim 26, further comprising:adding an aqueous medium to a blank well in the well plate, the blankwell being devoid of siRNA, wherein the aqueous medium optionallyincludes a polynucleotide carrier; adding cells to the blank well; andcomparing the effect of the first control siRNA on the cells in thefirst control well with the cells added to the blank well.
 30. A methodas in claim 26, further comprising: adding the aqueous medium includingthe polynucleotide carrier to a test well in the well plate, wherein thetest well includes a substantially dry gene silencing composition, thegene silencing composition having at least a first test siRNA whichsilences at least a first target gene, the gene silencing compositionbeing configured such that the at least first test siRNA is capable ofbeing solubilized or suspended in the transfection composition in anamount sufficient for transfecting cells in the well, wherein the genesilencing composition is characterized by the following: the at leastfirst test siRNA is not modified; the at least first test siRNA has astabilizing modification; the at least first test siRNA has amodification to limit off-targeting; the at least first test siRNA has aconjugate; the at least first test siRNA has a label; or the at leastfirst test siRNA has a hairpin structure.
 31. A method as in claim 26,further comprising: comparing the effect of the first control siRNA onthe cells with a known effect of the first control siRNA.
 32. A methodas in claim 25, wherein the polynucleotide carrier is a lipid.
 33. Amethod as in claim 24, further comprising maintaining the well plateunder conditions so that cell growth, cell division, transfection,and/or gene silencing occurs.
 34. A method as in claim 24, wherein thecells are added in an amount of about 2×10³ to about 3×10⁴ cells perabout 0.30 cm² to about 0.35 cm² of cell growth surface area.